HK40037022A - Portable molecular diagnostic device and methods for the detection of target viruses - Google Patents

Description

Portable molecular diagnostic device and method for detecting target virus

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application serial No. 62/583,789 entitled "portable molecular diagnostic test device with reverse transcription module" filed on 9.11.2017 and U.S. provisional application serial No. 62/594,905 entitled "portable molecular diagnostic test device and method for detecting target viruses" filed on 5.12.2017, each of which is incorporated herein by reference in its entirety.

Background

Embodiments described herein relate to devices and methods for molecular diagnostic testing. More particularly, embodiments described herein relate to disposable, stand-alone devices and methods for molecular diagnostic testing including reverse transcription capabilities.

Each year there are over one billion infections in the united states, many of which result in incorrect treatment due to inaccurate or delayed diagnostic outcomes. Many known point-of-care (POC) tests are poorly sensitive (30-70%), while more sensitive tests, such as those involving specific detection of nucleic acids or molecular tests associated with pathogenic targets, are only available in the laboratory. Therefore, molecular diagnostic tests are often performed in central laboratories. However, known devices and methods for conducting laboratory-based molecular diagnostic tests require trained personnel, a regulated infrastructure, and expensive high-throughput instruments. Known high throughput laboratory equipment typically processes many (96-384 and more) samples at a time, so central laboratory testing is often done in batches. Known methods for processing test samples typically include processing all samples collected over a period of one large-scale experiment (e.g., one day), resulting in a turnaround time of hours to days after sample collection. Furthermore, such known instruments and methods are designed to perform certain operations under the direction of a skilled technician who adds reagents, monitors the process and moves the sample step by step. Thus, although known laboratory tests and methods are very accurate, they often take a lot of time and are very expensive.

While some known laboratory-based molecular diagnostic test methods and equipment provide flexibility (e.g., the ability to test a variety of different indicators), such methods and equipment are not readily adaptable for point-of-care ("POC") applications or home use by untrained users. In particular, such known devices and methods use complex and include expensive and delicate components. Thus, use of such known laboratory-based methods and devices (e.g., POC or home applications) in decentralized locations may result in increased misuse, leading to inaccurate results or safety issues. For example, many known laboratory-based systems include delicate optical fibers and laser light sources, which are safety hazards for untrained users. Some known systems may also require the user to dispose of or be exposed to reagents that present a safety risk to an untrained user. For example, some known systems use relatively large amounts of reagents and/or require replenishment of reagents (e.g., within the instrument). In addition to being unsuitable for discrete use, these known systems are also unsuitable for long-term storage and transport. Long term preservation may be desirable, for example, to allow measured reserves for military applications, as part of the CDC national strategic reserves program, or other emergency preparation programs.

Furthermore, because of the flexibility provided by many known laboratory-based systems, such systems do not include prohibitions or mechanisms that prevent untrained users from completing certain actions beyond the correct sequence. For example, many known systems and methods include several different sample preparation operations, such as filtration, washing, lysis, and addition of sample preparation reagents to preserve target nucleic acids. If such operations are not performed in a predetermined order and/or within a predetermined time limit, the accuracy of the test may be compromised. Some known systems attempt to limit the complexity associated with sample preparation by limiting analysis to only "clean" samples. As a result, such systems cannot be a true end-to-end molecular diagnostic method, as detailed sample preparation still has to be performed by upstream processes.

While recent technological advances have enabled the development of "lab-on-a-chip" devices, such devices are often not optimized for point-of-care testing or home applications. For example, some known devices and methods require expensive or complex instrumentation to interface with the test cartridge, thereby increasing the likelihood of improper use. In addition, many known "lab-on-a-chip" devices amplify very small volumes of sample (e.g., less than 1 microliter) and are therefore unsuitable for analyzing multiple different metrics (e.g., 3-fold or 4-fold testing). Moreover, devices that produce such small sample volumes often include optical detection using photocells, charge coupled devices (CCD cameras), or the like, because sample volumes that are too small produce an output that can be read by the naked eye or by less sophisticated (and inexpensive) detectors.

Some known molecular diagnostic systems and methods facilitate the detection of viral pathogens by performing reverse transcription polymerase chain reaction (RT-PCR). While such methods may be useful for isolating and detecting viruses, they can be complex, thus making many known systems and methods unsuitable for decentralized and/or bedside use. For example, some known RT-PCR methods include additional steps to isolate and protect the target RNA from rapid degradation by ribonucleases (rnases). Inconsistencies when performing such methods may lead to inaccurate results due to variations in RNA degradation. Therefore, the known RT-PCR devices and methods are not suitable for use by untrained users.

Some known methods for detecting viruses, such as HIV, include detecting antibodies produced by the body in response to infection. Such antibody-based tests may be ineffective in identifying persons with acute and early HIV infection, as such tests are negative within weeks during the seronegative window after initial infection. In addition, while many known diagnostic tests are performed once to determine a preliminary diagnosis, some treatment regimens include repeated tests to assess the response of the treatment regimen. For example, many people diagnosed with HIV receive Antiretroviral (ARV) therapy. Although in many cases, the ARV treatment regimen reduces the HIV viral load in the blood to undetectable levels, some patients will experience a rebound in viral load levels due to issues of adhesion, development of drug resistance, and toxicity. Thus, the ARV treatment regimen also includes repeated viral load testing.

Accordingly, there is a need for improved devices and methods for molecular diagnostic testing. In particular, there is a need for improved devices and methods suitable for long-term storage. There is also a need for improved devices and methods that are easy to use and can be performed with minimal user input. There is also a need for improved devices and methods that can accommodate a wide range of samples (e.g., raw samples such as urine, saliva, and blood). There is also a need for improved devices and methods that include a reverse transcription module or otherwise allow for the detection of target RNA.

Disclosure of Invention

Described herein is a molecular diagnostic test device for amplifying nucleic acids within a sample and generating an indication of a target molecule (e.g., DNA or RNA) in the sample. In some embodiments, the method of detecting a target molecule comprises a "one-step" or "single button" actuation of the device. For example, in some embodiments, a method includes coupling a molecular diagnostic test device to a power source. A biological sample is transferred through an input opening into a sample preparation module within the molecular diagnostic test device. The molecular diagnostic test device is then actuated by only a single action to cause the molecular diagnostic test device to perform the following functions without further action by the user. First, the device heats the biological sample via the heater of the sample preparation module to lyse a portion of the biological sample to produce an input sample. Next, the device transfers the input sample to an amplification module within the molecular diagnostic test device. The device then heats the input sample within the reaction volume of the amplification module to amplify the nucleic acid molecules within the input sample, thereby generating an output solution containing the target amplicons. The device then reacts, within a detection module of the molecular diagnostic test device, each of (i) the output solution and (ii) a reagent formulated to produce a signal indicative of the presence of the target amplicon within the output solution. The detection module includes a detection surface configured to capture the target amplicons to generate the signal. The result associated with the signal is then read.

In some embodiments, molecular diagnostic test devices and related methods involve the use of multiple-use reagents (also known as buffers) to perform surface blocking and washing functions. In this way, the amount of reagent and the ease of use of the device may be increased, thereby facilitating bedside use, disposability of the device, and/or operation of the device in accordance with the CLIA-exempt (waived) method. In particular, in some embodiments, the multi-purpose reagent may include a blocking agent to reduce background signals associated with the attachment of undesirable particles during a detection event. By improving signal quality, such devices and methods may be adapted for use with limited sample preparation. In addition, the multi-purpose reagent may include a detergent that removes unbound components from the detection module. Such methods may include delivering quantities of the multi-purpose agent at different times depending on the desired function of the agent.

For example, in some embodiments, a method of detecting a nucleic acid using a molecular diagnostic test device includes transferring a first volume of a first reagent solution from a reagent module within the molecular diagnostic test device to a detection module within the molecular diagnostic test device at a first time. The detection module includes a detection surface configured to capture a target amplicon associated with the nucleic acid. The first reagent solution includes a blocking agent and a wash buffer. The first volume of the first reagent solution comprises an amount of the blocking solution sufficient to adsorb to a surface within the detection module. Transferring a sample solution comprising the target amplicon into the detection module at a second time such that the target amplicon is captured on the detection surface. After the second time, a second reagent solution is delivered to the detection module. The second reagent solution is formulated to cause generation of a signal indicative of the presence of the target amplicon within the sample solution. The method further includes transferring a second volume of the first reagent solution into the detection module after the second time. The second volume of the first reagent solution comprises a wash buffer in an amount sufficient to remove unbound components from at least one of the sample solution or the second reagent solution from the detection module.

In some embodiments, the method comprises lysing the original sample and performing a reverse transcription Polymerase Chain Reaction (PCR) on the lysed sample in the same environment. Stated another way, in some embodiments, the device includes a single lysis/RT-PCR module to facilitate methods that include lysing the original sample and performing rapid RT-PCR in a single chamber. Such methods can be performed in a manner that limits degradation of the target RNA after cleavage, thereby producing accurate results. Thus, such methods are suitable for execution by CLIA-exempt bedside devices.

For example, in some embodiments, a method of detecting nucleic acids includes mixing a reverse transcriptase enzyme with a biological sample within a sample preparation module to form a reverse transcription solution. Heating the reverse transcription solution within the sample preparation module to a first temperature within a lysis temperature range to release ribonucleic acid (RNA) molecules. Heating the reverse transcription solution to a second temperature within the reverse transcription temperature range within the same sample preparation module to produce complementary deoxyribonucleic acid (cDNA) molecules. The reverse transcription solution is then heated within the same sample preparation module to a third temperature above the inactivation temperature to cause inactivation of the reverse transcriptase. The method further includes transferring the reverse transcription solution to an amplification module where cDNA may be amplified for subsequent detection.

In some embodiments, a method of detecting a target RNA molecule using a disposable molecular diagnostic test device includes delivering an input sample to a reverse transcription module within a housing of the disposable molecular diagnostic test device. Heating the input sample within the reverse transcription module to generate target cDNA molecules associated with the target RNA molecules. Transferring the input sample from the reverse transcription module to an amplification module within the housing. The amplification module defines a reaction volume and includes a heater. The method further includes heating, by the heater, the input sample within at least a portion of the reaction volume to amplify the target cDNA molecules within the input sample, thereby producing an output solution comprising target amplicons. The method further comprises transmitting each of the following into the detection module: A) the output solution and B) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution, the detection module comprising a detection surface configured to retain the target amplicon to produce the signal. The disposable molecular diagnostic test device generates the signal when the viral load of the input sample is greater than 10 copies/ml.

Drawings

Fig. 1-3 are schematic illustrations of a molecular diagnostic test device according to one embodiment in a first, second, and third configuration, respectively.

FIG. 4 is a flow diagram of a method of detecting nucleic acids including a single actuation operation according to one embodiment.

Fig. 5 and 6 are schematic illustrations of a molecular diagnostic test device according to one embodiment in a first configuration and a second configuration, respectively.

FIG. 7 is a flow chart of a method of detecting nucleic acids according to one embodiment.

Fig. 8-11 are schematic illustrations of a molecular diagnostic test device using a multi-purpose reagent according to one embodiment in a first configuration, a second configuration, a third configuration, and a fourth configuration, respectively.

FIG. 12 is a flow chart of a method for detecting nucleic acids using a multipurpose reagent according to one embodiment.

FIG. 13 is a flowchart of a method of detecting nucleic acid including a reuse reagent according to one embodiment.

FIG. 14 is a graph illustrating signal generation resulting from an enzyme-linked reaction, according to one embodiment.

FIG. 15 is a schematic illustration of a molecular diagnostic test device according to one embodiment.

FIG. 16 is a schematic illustration of a portion of a molecular diagnostic test device including a single lysis and RT-PCR module, according to one embodiment.

FIGS. 17A-17C are graphs showing temperature versus time curves for various lysis and RT-PCR methods according to embodiments.

FIG. 18 is a flow diagram of a method of detecting nucleic acids comprising performing lysis and RT-PCR in a single environment, according to one embodiment.

FIG. 19 is a schematic illustration of a molecular diagnostic test device according to one embodiment.

FIGS. 20 and 21 are perspective and top views, respectively, of a molecular diagnostic test device according to one embodiment.

FIGS. 22 and 23 are exploded views of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 24 and 25 are front perspective views (FIG. 24) and rear perspective views (FIG. 25) of the molecular diagnostic test device shown in FIGS. 20 and 21, with the housing removed to show the modules therein.

Fig. 26 is an exploded perspective view of the housing assembly of the molecular diagnostic test device shown in fig. 20 and 21.

FIG. 27 is a bottom perspective view of the upper housing of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 28-30 are a front perspective view (FIG. 28), a rear perspective view (FIG. 29), and a bottom perspective view (FIG. 30) of the lid of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 31 and 32 are a top perspective view (FIG. 31) and a bottom perspective view (FIG. 32) of the flexible plate of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 33 and 34 are side cross-sectional views taken along line X-X in FIG. 21, showing the molecular diagnostic test device in a first (pre-actuation) configuration and a second (post-actuation) configuration, respectively.

FIGS. 35 and 36 are upper perspective views (FIG. 35) and bottom perspective views (FIG. 36) of the deformable support member of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 37 and 38 are perspective (FIG. 37) and top (FIG. 38) views of the sample preparation (or fractionation) module of the molecular diagnostic test device shown in FIGS. 20 and 21.

Fig. 39 and 40 are a cross-sectional view (fig. 39) and an exploded view (fig. 40) of the sample preparation module shown in fig. 37 and 38.

FIG. 41 is a cross-sectional view of the mixing assembly of the sample preparation module shown in FIGS. 37 and 38 taken along line X-X in FIG. 38.

FIG. 42 is a top view of the flow member of the amplification module of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIG. 43 is an exploded view of the detection module of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 44 and 45 are a top perspective view (FIG. 44) and a bottom perspective view (FIG. 45) of a reagent module of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIG. 46 is a front perspective view of the rotary valve assembly of the molecular diagnostic test device shown in FIGS. 20 and 21.

FIGS. 47-52 are front views of the rotary valve assembly shown in FIG. 46, with the vent housing being "transparent" to show the valve flaps of each of the six different operating configurations.

Fig. 53A-53C are perspective views of the molecular diagnostic device shown in fig. 20 and 21 at various stages of operation, according to one embodiment.

Detailed Description

In some embodiments, the device is configured for a disposable, portable, single use, inexpensive means of molecular diagnosis. The apparatus may include one or more modules configured to perform high quality molecular diagnostic tests including, but not limited to, sample preparation, nucleic acid amplification (e.g., by polymerase chain reaction, isothermal amplification, etc.), and detection. In some embodiments, sample preparation may be performed by isolating the target pathogen/entity and removing undesired amplification (e.g., PCR) inhibitors. The target entity can subsequently be cleaved to release the target nucleic acid for amplification. The target nucleic acid in the target entity can be amplified using a polymerase to undergo temperature cycling or by isothermal incubation to produce a large number of copies of the target nucleic acid sequence for detection.

In some embodiments, the devices described herein are stand-alone devices that include all the necessary substances, mechanisms, and components to perform any of the molecular diagnostic tests described herein. Such a freestanding device does not require any external instrument to manipulate the biological sample, only requires connection to a power source (e.g., to an a/C power source, coupled to a battery, etc.) to accomplish the methods described herein. For example, the devices described herein do not require any external instruments to heat the sample, agitate or mix the sample, pump (or move) fluids within the flow member, and the like. Rather, the embodiments described herein are self-contained and upon addition of a biological sample and coupling to a power source, the device can be actuated to perform the molecular diagnostic tests described herein. In some embodiments, the method of actuating the device may be such that the device is a CLIA-exempt device and/or may operate according to a CLIA-exempt method.

In some embodiments, the method of detecting a target molecule comprises a "one-step" or "single button" actuation of the device. For example, in some embodiments, a method comprises coupling the molecular diagnostic test device to a power source. A biological sample is transferred through an input opening into a sample preparation module within the molecular diagnostic test device. The molecular diagnostic test device is then actuated by only a single action to cause the molecular diagnostic test device to perform the following functions without further action by the user. First, the device heats the biological sample via the heater of the sample preparation module to lyse a portion of the biological sample to produce an input sample. Next, the device transfers the input sample to an amplification module within the molecular diagnostic test device. The device then heats the input sample within the reaction volume of the amplification module to amplify the nucleic acid molecules within the input sample, thereby generating an output solution containing the target amplicons. The device then reacts, within the detection module of the molecular diagnostic test device, each of: (i) the output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution. The detection module includes a detection surface configured to capture the target amplicons to generate the signal. The result associated with the signal is then read.

In some embodiments, the apparatus may include a cover (also referred to as a cover) that functions to both cover the input sample port and actuate one or more mechanisms of the device when the cover is closed. In this way, a single action of closing the lid also actuates all aspects of the device, thereby simplifying device actuation and methods. In particular, in some embodiments, a method of detecting nucleic acids includes coupling a molecular diagnostic test device to a power source and delivering a biological sample through an input opening into a sample preparation module within the molecular diagnostic test device. The order of these operations does not matter. To actuate the device, the input opening is covered with a cover that is connected to the molecular diagnostic test device. In response to the mere act of covering, the device then performs the following functions without further action by the user. First, the device heats the biological sample via the heater of the sample preparation module to lyse a portion of the biological sample to produce an input sample. Next, the device transfers the input sample to an amplification module within the molecular diagnostic test device. The device then heats the input sample within the reaction volume of the amplification module to amplify the nucleic acids within the input sample, thereby generating an output solution containing the target amplicons. The device then reacts, within the detection module of the molecular diagnostic test device, each of: (i) the output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution. The detection module includes a detection surface configured to capture the target amplicons to generate the signal. The result associated with the signal is then read.

In some embodiments, an apparatus includes a housing, a sample preparation module within the housing, a reagent module within the housing, a detection module, and a lid removably coupled to the housing. The sample preparation module defines a sample input volume that receives a biological sample and an input opening through which the sample input volume is accessible. The sample preparation module includes a heater configured to heat the biological sample to produce an input solution. The reagent module includes a reagent container comprising a detection reagent configured to facilitate generation of a signal indicative of the presence of a target amplicon from the input solution. The detection reagent is sealed within the reagent container. The seal may be, for example, a foil seal that maintains the shelf life of the reagent and prevents leakage of the reagent. The detection module includes a detection surface configured to capture the target amplicons from the input solution. The detection module is in fluid communication with the reagent module such that the signal is generated in response to the reagent being delivered into the detection module. The lid includes a sealing portion, a switching portion, and a reagent actuator. The cover moves relative to the housing between a first cover position and a second cover position. The input opening is exposed when the lid is in the first lid position and the sealing portion of the lid covers the input opening when the lid is in the second lid position. When the lid is moved from the first lid position to the second lid position: A) the switch portion actuates a switch to provide power to the heater, and B) the reagent actuator causes the reagent to be released from the sealed reagent container.

In some embodiments, the device further comprises an amplification module within the housing, the amplification module configured to receive the input solution from the sample preparation module. The amplification module is configured to heat the input solution to amplify nucleic acids within the input solution, thereby generating a detection solution comprising the target amplicons.

In some embodiments, the lid comprises a detent that irreversibly engages at least one of the housing, the sample preparation module, or the reagent module, thereby retaining the lid at the second lid position. In this way, the molecular diagnostic apparatus is configured to be irreversibly used. That is, this configuration prevents reuse of the device or prevents subsequent attempts to replenish the biological sample after the device has been actuated.

In some embodiments, the reagent module comprises a reagent housing and a piercer. The reagent housing defines a reagent reservoir into which the reagent is released from the sealed reagent container when the piercer pierces a portion of the reagent container. The reagent actuator includes a protrusion that applies a force to cause the piercer to pierce the portion of the reagent container when the lid is moved from the first lid position to the second lid position. In some embodiments, the apparatus comprises a deformable support member configured to hold the piercer and/or the reagent container in a spaced apart position. The deformable support member is configured to deform in response to a force applied when the cover is moved to the second position to move the piercer and/or the reagent container into contact with one another.

In some embodiments, an apparatus includes a housing of a molecular diagnostic device and a reagent module within the housing. The reagent module includes a reagent housing, a reagent container containing a reagent sealed therein, a piercer, and a deformable support member. The reagent housing defines a reagent reservoir into which the reagent is released from the reagent container when the piercer pierces a portion of the reagent container. The deformable support member includes a sealing portion and a connecting portion. The seal is connected to the reagent housing to fluidly isolate the reagent reservoir. The connection portion is connected to at least one of the piercer or the reagent container. The deformable support member is configured to deform from a first configuration to a second configuration in response to an actuation force applied to the deformable support member. The deformable support member holds the piercer spaced apart from the portion of the reagent container when the deformable support member is in the first configuration. The piercer pierces the portion of the reagent container when the deformable support member is in the second configuration.

In some embodiments, the agent is one of a first agent or a second agent. The first reagent is formulated to bind to the target molecule in response to the first reagent being delivered into the detection module, and the second reagent is formulated to produce the signal when catalyzed by the first reagent. The second agent can be, for example, a precipitation substrate formulated to produce insoluble colored particles when the second agent is contacted with the first agent.

In some embodiments, the reagent is a first reagent and is one of a catalytic reagent configured to bind to the target molecule in response to the first reagent being delivered into the detection module or a precipitating reagent configured to generate the signal when catalyzed by the catalytic reagent. The reagent module includes a second reagent container containing a solution including a wash buffer and a blocking buffer configured to reduce attachment of the target amplicon or other molecule within the detection module. The connecting portion of the deformable support member is connected to at least one of a second piercer or the second reagent container. The deformable support member holds the second piercer away from the second reagent container when the deformable support member is in the first configuration. The second piercer pierces the second reagent container when the deformable support member is in the second configuration.

In some embodiments, the molecular diagnostic test devices and related methods involve the use of multi-purpose reagents (also referred to as buffers) to perform both surface blocking and washing functions. In this way, the amount of reagent and the ease of the device may be increased, thereby facilitating bedside use, disposability of the device, and/or device operation in accordance with CLIA-exempt methods. In particular, in some embodiments, the multi-purpose reagent may include a blocking agent to reduce background signals associated with the attachment of undesirable particles during a detection event. By improving signal quality, such devices and methods may be adapted for use with limited sample preparation. In addition, the multi-purpose reagent may include a detergent that removes unbound components from the detection module. Such methods may include delivering quantities of the multi-purpose agent at different times depending on the desired function of the agent.

For example, in some embodiments, a method of detecting a nucleic acid using a molecular diagnostic test device includes transferring a first volume of a first reagent solution from a reagent module within the molecular diagnostic test device to a detection module within the molecular diagnostic test device at a first time. The detection module includes a detection surface configured to capture a target amplicon associated with the nucleic acid. The first reagent solution includes a blocking agent and a wash buffer. The first volume of the first reagent solution comprises an amount of the blocking solution sufficient to adsorb to a surface within the detection module. Transferring a sample solution comprising the target amplicon into the detection module at a second time such that the target amplicon is captured on the detection surface. After the second time, a second reagent solution is delivered to the detection module. The second reagent solution is formulated to cause generation of a signal indicative of the presence of the target amplicon within the sample solution. The method further includes transferring a second volume of the first reagent solution into the detection module after the second time. The second volume of the first reagent solution comprises the wash buffer in an amount sufficient to remove unbound components from at least one of the sample solution or the second reagent solution from the detection module. In some embodiments, the first reagent solution comprises 0.02% -5% bovine serum albumin and 0.05% -10% of the detergent.

In some embodiments, the method of detecting nucleic acids using a molecular diagnostic test device comprises reusing a multi-purpose reagent. In particular, the reagent may be used at a first time to perform a blocking function and then may be transported through the detection module at a second time to perform a washing function. This configuration and method enables the containment of fewer reagents within a molecular diagnostic test device, thereby facilitating a more efficient, lower cost single use, stand-alone device. In particular, in some embodiments, a method of detecting nucleic acids using a molecular diagnostic test device includes delivering a biological sample through an input opening into a sample preparation module within the molecular diagnostic test device. The device is then actuated to cause the device to perform the following functions. First, the device transfers a first volume of reagent solution from a reagent module within the molecular diagnostic test device to a detection module comprising a detection surface configured to capture target amplicons associated with the nucleic acids. The reagent solution includes a blocking agent and a wash buffer, the blocking agent being formulated to adsorb to a surface within the detection module. The device then transfers the first volume of the reagent solution from the detection module back to the reagent module. An output solution is then generated from the biological sample that includes the target amplicon associated with the nucleic acid. This may be performed by any of the sample preparation modules or amplification modules described herein. The output solution is then conveyed into the detection module such that the target amplicons are captured on the detection surface. The device then transfers a second volume of the reagent solution from the reagent module into the detection module, thereby removing unbound components from the output solution from the detection module. The results associated with the target amplicons captured on the detection surface are then read.

In some embodiments, the method comprises lysing the original sample and performing a reverse transcription Polymerase Chain Reaction (PCR) on the lysed sample in the same environment. Stated another way, in some embodiments, the device includes a single lysis/RT-PCR module to facilitate methods that include lysing the original sample and performing rapid RT-PCR in a single chamber. Such methods can be performed in a manner that limits degradation of the target RNA after cleavage, thereby producing accurate results. Thus, such methods are suitable for execution by CLIA-exempt bedside devices.

For example, in some embodiments, a method of detecting nucleic acids includes mixing a reverse transcriptase enzyme with a biological sample within a sample preparation module to form a reverse transcription solution. Within the sample preparation module, the reverse transcription solution is heated to a first temperature within a lysis temperature range to release ribonucleic acid (RNA) molecules. Within the same sample preparation module, the reverse transcription solution is heated to a second temperature within the reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules. The reverse transcription solution is then heated to a third temperature above the inactivation temperature within the same sample preparation module to cause inactivation of the reverse transcriptase. The method further includes transferring the reverse transcription solution to an amplification module where cDNA may be amplified for subsequent detection.

In some embodiments, a method of detecting a nucleic acid includes mixing a reverse transcriptase enzyme with a biological sample within a sample preparation module to form a reverse transcription solution. Within the sample preparation module, the reverse transcription solution is heated to a first temperature within a lysis temperature range to release ribonucleic acid (RNA) molecules. Within the same sample preparation module, the reverse transcription solution is heated to a second temperature within the reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules. Said heating to said first temperature and said heating to said second temperature are performed continuously such that said cDNA is produced in less than 1 minute of releasing said RNA molecule.

In some embodiments, a method of detecting a target RNA molecule using a disposable molecular diagnostic test device includes delivering an input sample to a reverse transcription module within a housing of the disposable molecular diagnostic test device. Heating the input sample within the reverse transcription module to generate target cDNA molecules associated with the target RNA molecules. Transferring the input sample from the reverse transcription module to an amplification module within the housing. The amplification module defines a reaction volume and includes a heater. The method further includes heating, by the heater, the input sample within at least a portion of the reaction volume to amplify the target cDNA molecules within the input sample, thereby producing an output solution comprising target amplicons. The method further comprises transmitting each of the following into the detection module: A) the output solution and B) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution, the detection module comprising a detection surface configured to retain the target amplicon to produce the signal. The disposable molecular diagnostic test device generates the signal when the viral load of the input sample is greater than 1000 copies/ml. In other embodiments, the disposable molecular diagnostic test device may generate the signal when the viral load of the input sample is greater than 100 copies/ml. In still other embodiments, the disposable molecular diagnostic test device may generate the signal when the viral load of the input sample is greater than 10 copies/ml.

In some embodiments, the device comprises a housing, a sample preparation module, a reverse transcription module, and an amplification module, each module within the housing. The sample preparation module defines an input reservoir configured to receive a blood sample. The sample preparation module is configured to separate a plasma sample from the blood sample, the plasma sample comprising a target RNA molecule. The reverse transcription module is configured to heat the plasma sample to generate target cDNA molecules associated with the target RNA molecules, thereby generating an amplification solution. The amplification module includes a flow member and a heater. The flow member defines a reaction volume configured to receive the amplification solution. The heater is configured to deliver thermal energy into the reaction volume to amplify the target cDNA molecules within the amplification solution to produce an output solution comprising target amplicons.

In some embodiments, a method of detecting a target RNA molecule using a molecular diagnostic test device includes first transferring a biological sample into a sample preparation module within a disposable molecular diagnostic test device. The device is then actuated to cause the device to perform the following functions. The device heats the biological sample within a reverse transcription portion of the sample preparation module to produce target DNA molecules associated with the target RNA molecules, thereby producing an amplified sample. Mixing the target cDNA with a primer composition associated with a plurality of target sequences of the target cDNA molecule. The amplified sample is then transferred to an amplification module within the device, and the amplified sample is then heated to amplify each of the plurality of target sequences of the target cDNA molecules within the amplified sample, thereby generating an output solution comprising a plurality of target amplicons. The device then transmits each of the following to the detection module: A) the output solution and B) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution. The detection module includes a detection surface configured to retain the plurality of target amplicons within a single region to generate the signal. The method further comprises reading out the signal from the detection surface.

As used in this specification and the appended claims, the term "reagent" includes any substance used in conjunction with any of the reactions described herein. For example, the reagents may include elution buffers, PCR reagents, enzymes, substrates, wash solutions, blocking solutions, and the like. The reagent may comprise a mixture of one or more components. The reagent may include such components regardless of their physical state (e.g., solid, liquid, or gas). In addition, the reagent may include a plurality of components that may be included in a substance in a mixed state, an unmixed state, and/or a partially mixed state. The agent may include both active and inert ingredients. Thus, as used herein, an agent may include inactive and/or inert ingredients such as water, colorants, and the like.

The terms "nucleic acid molecule," "nucleic acid," or "polynucleotide" may be used interchangeably herein and may refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including known analogs or combinations thereof, unless otherwise specified. The nucleic acid molecules to be commented on herein may be obtained from any source of nucleic acid. The nucleic acid molecule may be single-stranded or double-stranded. In some cases, the nucleic acid molecule is DNA. The DNA may be mitochondrial DNA, complementary DNA (cDNA) or genomic DNA. In some cases, the nucleic acid molecule is genomic dna (gdna). The DNA may be plasmid DNA, cosmid DNA, Bacterial Artificial Chromosome (BAC), or Yeast Artificial Chromosome (YAC). The DNA may be from one or more chromosomes. For example, if the DNA is from a human, the DNA may be from one or more of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, X, or Y. In some cases, the nucleic acid molecule is RNA, including, but not limited to, mRNA, tRNA, snRNA, rRNA, retrovirus, small non-coding RNA, microrna, multimeric RNA (polysomal RNA), pre-mRNA, intronic RNA, viral RNA, cell free RNA, and fragments thereof. Non-coding RNAs or ncrnas may include snornas, micrornas, sirnas, pirnas, and long nc RNAs. The source of the nucleic acids used in the devices, methods, and compositions described herein can be a sample comprising the nucleic acids.

Unless otherwise indicated, the terms device, diagnostic system, diagnostic test system, test apparatus, and variations thereof may be used interchangeably.

The methods described herein can be performed on any suitable molecular diagnostic device, such as any of the diagnostic devices shown and described herein or in international patent publication No. WO2016/109691 entitled "device and method for molecular diagnostic testing", international patent publication No. WO2017/185067 entitled "printed circuit board heater for amplification module", international patent publication No. WO2018/005710 entitled "device and method for detecting molecules using a flow cell", and international patent publication No. WO2018/005870 entitled "device and method for nucleic acid extraction" (the entire contents of each of the aforementioned patents are incorporated herein by reference).

Fig. 1-3 are schematic illustrations of a molecular diagnostic test device 1000 (also referred to as a "test device" or "device") according to one embodiment. According to any of the methods described herein, the testing device 1000 is configured to manipulate a biological sample to produce one or more output signals associated with a target cell. In some implementations, the testing device 1000 may be an integrated device suitable for use within a bedside facility (e.g., a doctor's office, pharmacy, etc.), a distributed testing facility, or in the home of a user. Similarly, in some embodiments, the modules of the devices described below are housed within a single housing so that the testing device can operate entirely without any additional instrumentation, docking stations (dockingstation), or the like. Further, in some embodiments, the device 1000 may have a size, shape, and/or weight such that the device 1000 may be carried, held, used, and/or operated in a user's hand (i.e., it may be a "handheld" device). In some embodiments, the test device 1000 may be a self-contained single-use device.

To facilitate ease of use, in addition to inputting a biological sample and connecting the device to a power source, the device 1000 is configured to be actuated in a single step or action. The "single button" actuation reduces the complexity of the operating steps, thereby making the device and method suitable for use by untrained users. As described below, the device does not require the operation of multiple different actuators (or buttons) to perform sample preparation, does not require vibration or external agitation, and does not require a complex "signal reading" step.

In some embodiments, the device 1000 (as well as any of the devices shown and described herein) may be a CLIA-exempt device and/or may operate according to a CLIA-exempt method. Similarly, in some embodiments, device 1000 (as well as any of the other devices shown and described herein) is configured to operate in a sufficiently simple manner and can produce sufficiently accurate results to create a limited likelihood of misuse and/or a limited risk of harm when improperly used. In some embodiments, the device 1000 (as well as any of the other devices shown and described herein) may be operated by a user with little (or no) scientific training, in accordance with methods that require little user judgment and/or in which certain operational steps are easily controlled and/or automatically controlled. In some embodiments, the molecular diagnostic test device 1000 may be configured for long-term storage in a manner that creates a limited likelihood of improper use (reagent damage, reagent expiration, reagent leakage, etc.). In some embodiments, the molecular diagnostic test device 1000 is configured to be stored for up to about 36 months, up to about 32 months, up to about 26 months, up to about 24 months, up to about 20 months, up to about 18 months, up to 12 months, up to 6 months, or up to any time therebetween.

The testing device 1000 includes a housing 1001, an actuator 1050, a sample preparation module 1200 (also referred to as a sample staging module), an amplification module 1600, and a detection module 1800. In some embodiments, the testing device 1000 can include any other component or module described herein, such as, for example, a reagent module containing on-board reagents (e.g., reagent module 6700), a rotary valve (e.g., for controlling the flow of reagents and/or samples, such as valve 6300), or a fluid transfer module (e.g., fluid transfer module 6400). The housing 1001 may be any structure in which the sample preparation module 1200 or other components are housed (or partially housed) to form an integrated device for sample preparation and/or molecular testing. The housing 1001 may be a unitary constructed housing or may include a plurality of separately constructed components that are subsequently joined together to form the housing 1001. As shown in fig. 2, the housing defines an input opening 1021 through which a biological sample S1 may be transferred into the sample preparation module 1200.

The sample preparation module 1200 includes a heater 1230 and is configured to operate the biological sample S1 for further diagnostic testing. For example, in some embodiments, the sample preparation module 1200 may extract nucleic acid molecules from the biological sample S1 and may generate an output solution S2 (see fig. 3) that is delivered into the amplification module 1600. The sample preparation module 1200 can include any other components described herein, such as, for example, a heater for lysis, a chamber within which RT-PCR can be performed, and/or a deactivation chamber (see, e.g., lysis housing 6201).

The amplification module 1600 defines an internal volume (e.g., a reaction chamber or reaction volume) and includes a heater 1630. The reaction volume may be a single volume or a series of volumes within which the input solution S2 (i.e., the solution containing the nucleic acids extracted from the biological sample S1) may flow and/or be held to amplify the target nucleic acid molecules therein, thereby producing the output detection solution S3 containing the target amplicons to be detected. In some embodiments, the reaction volume comprises a serpentine flow path such that the flow path intersects the heater 1630 at multiple locations. In this manner, the amplification module 1600 can perform a "flow-through" amplification reaction in which the input solution S2 flows through multiple different temperature zones.

The heater 1630 may be any suitable heater or group of heaters that can heat the input solution S2 to perform any amplification operation as described herein. In some embodiments, heater 1630 may establish a plurality of temperature bands through which the prepared solution may flow, and/or heater 1630 may define a desired number of amplification cycles to ensure a desired test sensitivity (e.g., at least 30 cycles, at least 34 cycles, at least 36 cycles, at least 38 cycles, or at least 40 cycles). The heater 1630 (as well as any of the heaters described herein) can have any suitable design. For example, in some embodiments, the heater 1630 can be a resistive heater, a thermoelectric device (e.g., a Peltier device), or the like.

In some embodiments, the amplification module 1600 (or any of the amplification modules described herein) can be similar to the amplification module shown and described in U.S. patent publication No. 2017/0304829 entitled "printed circuit board heater for amplification module" (which application is incorporated by reference herein in its entirety). In other embodiments, the amplification module 1600 (or any of the amplification modules described herein) can be similar to the amplification module shown and described in international patent publication No. WO2016/109691, entitled "apparatus and methods for molecular diagnostic testing" (which application is incorporated herein by reference in its entirety). Although amplification module 1600 is generally described as performing thermal cycling operations on input solution S2, in other embodiments, amplification module 1600 (and any of the amplification modules described herein) may perform any suitable thermal reaction to amplify nucleic acids within the solution. In some embodiments, the amplification module 1600 (and any amplification module described herein) can perform any suitable type of isothermal amplification process, including, for example, loop-mediated isothermal amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA) useful for detecting a target RNA molecule, Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), reticulo-branch amplification (RAM), or any other type of isothermal process.

The detection module 1800 is configured to react the output solution S3 from the amplification module 1600 with one or more reagents to generate a signal (or output) OP1, indicating the presence or absence of a target organism in the biological sample S1. In particular, detection module 1800 defines a detection channel and includes a detection surface 1821 within the detection channel. The detection channel is in fluid communication with (or placed in fluid communication with) the amplification module 1600. In this manner, the output solution S3 containing the target amplicons can be conveyed into the detection channel and past the detection surface 1821. In addition, as shown in fig. 3, a reagent R configured to generate, catalyze, or otherwise facilitate generation of a signal indicative of the presence of a target amplicon can be delivered into the detection channel and past the detection surface 1821. The detection surface 1821 includes a series of capture probes to which target amplicons can bind as the output solution S3 flows across the detection surface 1821. The capture probe may be any suitable probe of the type described herein that is formulated to capture or bind the target amplicon.

The molecular diagnostic test device 1000 (as well as any of the molecular diagnostic test devices described herein) can perform any of the "one-touch" actuation methods described herein. For example, FIG. 4 is a flow chart of a method 10 for detecting nucleic acids according to one embodiment. Although the method 10 is described as being performed on the apparatus 1000, in other embodiments, the method 10 may be performed on any suitable apparatus, such as the apparatus 6000 described below. The method 10 includes coupling a molecular diagnostic test device to a power source at 12. Referring to fig. 1 and 2, power supply 1905 can be coupled to a terminal 1940 of the device, as indicated by arrow AA. The power supply 1905 can be any suitable power source, such as an alternating current (A/C) power source, a direct current (D/C) power source (e.g., a battery), a fuel cell, and the like. In some embodiments, power source 1905 can be an a/C power source, and connecting can include plugging the device into an electrical outlet using a power cord. In other embodiments, power source 1905 can be a D/C power source, and connecting can include coupling a battery to terminals 1940 of the device. In still other embodiments, the power source 1905 can be a D/C power source disposed within the housing of the device, and the coupling can include removing an electrically insulating element from between the power source and the rest of the electronic controls (not shown in fig. 1-3) of the device.

The biological sample is transferred at 13 through the input opening to a sample preparation module within the molecular diagnostic test device. Referring to fig. 2, in some embodiments, the biological sample S1 may be transferred into the device by a sample transfer device 1110. The sample transfer device 1110 may be any suitable device, such as a pipette or other mechanism configured to aspirate or extract a sample S1 from a sample cup, container, or the like and then transfer a desired amount of the sample through the opening 1021. Biological sample S1 may be any suitable sample, such as, for example, blood, urine, a male urethral specimen, a vaginal specimen, a cervical swab specimen, a nasal swab specimen, a pharyngeal swab specimen, a rectal swab specimen, or any other biological sample described herein. Thus, in some embodiments, the biological sample S1 may be a "raw" (or unprocessed) sample.

The molecular diagnostic test device is then actuated at 14 with only a single action, causing the molecular diagnostic test device to perform a series of operations without any further user input. In other words, the molecular diagnostic test device is actuated by only a "single button", as shown by arrow BB and actuator 1050 in fig. 2. Although actuator 1050 is shown as a push button actuator, the "single action" in operation 14 may be performed by any suitable mechanism. For example, in some embodiments, the device may include a sliding actuator that actuates the device when the actuator slides relative to the device housing. In other embodiments, the device may include a rotary actuator or an actuator that is removed (e.g., torn off) from the device to initiate operation of the device. For example, in some embodiments, the actuator may be a peel strip that covers a window through which signals are read. In still other embodiments, the actuator can be a cover, similar to covers 2050 or 6050, that also actuates aspects of the device when closed.

Upon actuation by a "single button," the molecular diagnostic test device may perform any of the methods described herein. Specifically, at 14A, the device can heat a biological sample via a heater of a sample preparation module to lyse a portion of the biological sample, thereby generating an input sample. Referring to fig. 3, the biological sample S1 may be heated by a heater 1230 and the resulting lysed sample (i.e., input sample S2) may be transported toward the amplification module 1600. Although device 1000 does not show any additional sample preparation, in other embodiments, the biological sample may be filtered, separated, eluted, subjected to an enzyme deactivation heating operation, or the like to produce a suitable input sample S2. However, in other embodiments, the method need not include any filtration or other separation techniques.

The input sample is then transferred to an amplification module within the molecular diagnostic test device at 14B. Referring to fig. 3, the amplification module defines a reaction volume, as described above. Thus, at 14C, the input sample is heated within the reaction volume to amplify the nucleic acids within the input sample, thereby producing an output solution comprising the target amplicons. The input solution can be amplified by using any suitable technique (e.g., PCR, isothermal amplification, etc.), as described herein.

After amplification, the device then reacts, at 14D, within the detection module of the molecular diagnostic test device, each of the following: (i) the output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution. As shown in fig. 3, detection module 1800 includes a detection surface 1821 configured to capture target amplicons to generate output signal OP 1. The output signal OP1 may be any suitable signal. In some embodiments, the output signal OP1 may be a colorimetric signal indicative of the presence of bound amplicons: if the target pathogen, target amplicon, and/or target organism is present, a colored product is formed, and if the target pathogen, target amplicon, and/or target organism is not present, a colored product is not formed.

Reagent R may be any suitable reagent type described herein and may be introduced into detection module 1800 by any suitable mechanism. For example, in some embodiments, the reagent may be a catalyst formulated to bind to a target molecule in response to being delivered into the detection module 1800. In other embodiments, the reagent may be configured to generate a signal when catalyzed by another reagent already present in the detection module 1600. In some embodiments, the agent may be a precipitation substrate formulated to produce insoluble colored particles when the agent is contacted with a catalyst. The reagent R may be present in the detection module before the device is actuated, or alternatively, the reagent R may be transferred to the detection module as a result of device actuation. For example, in some embodiments, the device can include an on-board reagent module (e.g., reagent module 6700), and when the device is actuated, the device can release reagent into a shunt (harness) or "sump" for subsequent use during operation. In some embodiments, the device may comprise a fluid transfer device or pump, similar to the fluid transfer device 6400 described herein.

The method further comprises reading out a result related to the signal at 15. In some embodiments, the readout can include visual inspection of the device and detection surface 1821 for colorimetric signals. In other embodiments, the signal OP1 generated by detection surface 1821 need not be visible to the naked eye. For example, in some embodiments, the readout may include scanning or otherwise receiving the signal OP1 using an auxiliary device, such as a mobile computing device. In still other embodiments, the reading of the results can include indirectly reading a secondary signal that conveys a result related to (or descriptive of) the primary output from detection surface 1821.

In some embodiments, method 10 optionally comprises discarding the molecular testing device after the readout. In some embodiments, the amount of sample and reagents may be such that the device can be disposed of by standard, unconstrained waste treatment procedures. In other embodiments, the discarding comprises disposing of the used device through standard medical waste disposal procedures.

In some embodiments, method 10 optionally comprises storing the molecular diagnostic test device comprising any reagents sealed therein for at least 6 months prior to use.

Although method 10 shows the operation of coupling the device to a power source to occur before the biological sample is transferred to the device, in other embodiments, any of the steps of method 10 (or any of the methods described herein) may be performed in any order or may be performed simultaneously. For example, in some embodiments, the biological sample S1 may be first transferred into the device, the device may be actuated (via actuator 1050), and then after actuation, the device may be inserted into a receptacle to provide a/C power to the device.

In some embodiments, the apparatus may include a cover (also referred to as a cover) that functions to cover both the input opening and the one or more mechanisms that actuate the device when the cover is closed. In this way, a single action of closing the lid also actuates all aspects of the device, thereby simplifying device actuation and methods.

For example, fig. 5 and 6 are schematic illustrations of a molecular diagnostic test device 2000 (also referred to as a "test device" or "device") according to one embodiment. The testing device 2000 is configured to manipulate a biological sample to produce one or more output signals associated with a target cell according to any of the methods described herein. In some embodiments, the testing device 2000 may be an integrated device suitable for use within a bedside facility (e.g., a doctor's office, pharmacy, etc.), a distributed testing facility, or in a user's home. Similarly, in some embodiments, the modules of the devices described below are housed within a single housing so that the testing device can operate entirely without any additional instrumentation, docking stations, or the like. Further, in some embodiments, the device 2000 may have a size, shape, and/or weight such that the device 2000 may be carried, held, used, and/or operated in a user's hand (i.e., it may be a "handheld" device). In some embodiments, the testing device 2000 may be a self-contained single-use device.

In some embodiments, the device 2000 (as well as any of the devices shown and described herein) may be a CLIA-exempt device and/or may operate according to a CLIA-exempt method. Similarly, in some embodiments, the device 2000 (as well as any of the other devices shown and described herein) is configured to operate in a sufficiently simple manner and can produce sufficiently accurate results, thereby creating a limited likelihood of misuse and/or a limited risk of harm when improperly used. In some embodiments, the device 2000 (as well as any of the other devices shown and described herein) may be operated by a user with little (or no) scientific training, in accordance with methods that require little user judgment and/or in which certain operational steps are easily controlled and/or automatically controlled. In some embodiments, the molecular diagnostic test device 2000 may be configured for long-term storage in a manner that creates a limited likelihood of improper use (reagent damage, reagent expiration, reagent leakage, etc.). In some embodiments, the molecular diagnostic test device 2000 is configured to be stored for up to about 36 months, up to about 32 months, up to about 26 months, up to about 24 months, up to about 20 months, up to about 18 months, up to 12 months, up to 6 months, or any value therebetween.

The testing device 2000 includes a housing 2001, a lid 2050, a sample preparation module 2200 (also referred to as a sample staging module), a reagent module 2700, a detection module 2800, and an electronic control module 2950. In some embodiments, the testing device 2000 can include any other component or module described herein, such as, for example, an amplification module (e.g., amplification module 1600 or 6600), a rotary valve (e.g., to control the flow of reagents and/or samples, such as valve 6300), or a fluid transfer module (e.g., fluid transfer module 6400). The housing 2001 may be any structure in which the sample preparation module 2200 or other components are housed (or partially housed) to form an integrated device for sample preparation and/or molecular testing.

The sample preparation module 2200 defines a sample input volume 2211 that receives the biological sample S1 and an input opening 2212 through which the biological sample S1 may be transferred into the sample preparation module 2200. Sample preparation module 2200 includes heater 2230 and is configured to operate biological sample S1 for further diagnostic testing. For example, in some embodiments, the sample preparation module 2200 may extract nucleic acid molecules from the biological sample S1 and may generate an input solution S2 (see fig. 6), which input solution S2 is optionally transported into an amplification module (not shown), or into a detection module 2800. Sample preparation module 2200 can include any other component described herein, such as, for example, a heater for lysis, a chamber within which RT-PCR can be performed, and/or a deactivation chamber (see, e.g., lysis housing 6201).

The reagent module 2700 is disposed within the housing 2001 and includes a reagent reservoir 2701, a plunger 2755, and a reagent reservoir 2730. Reagent module 2700 provides on-board storage of reagent R for use in connection with the molecular diagnostic tests described herein. Reagent R may be any reagent type shown and described herein. For example, in some embodiments, reagent R may be a detection reagent formulated to facilitate production of a signal indicative of the presence of a target amplicon from input solution S2. Thus, reagent R can be formulated to include a binding moiety and any suitable enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase. In some embodiments, the HRP enzyme has been conjugated to a streptavidin molecule. In some embodiments, the reagent R may be a substrate that when catalyzed generates a colored molecule. In other embodiments, reagent R may be a wash buffer or blocking agent, each of which may contribute to the generation of the signal (e.g., by reducing spurious outputs), as described herein.

Prior to actuation, reagent R is sealed within reagent reservoir 2701. In some embodiments, reagent R may be sealed by frangible portion 2713 of reagent container 2701. In other embodiments, the reagent container 2701 may include any suitable sealing mechanism. By sealing reagent R within reagent reservoir 2701, device 2000 may be suitable for long-term storage, and reagent R may be protected from degradation, and the like. Reagent plunger 2755 includes a piercer 2754. As shown in fig. 5, prior to actuation, the piercer is spaced from the frangible portion 2713, thereby maintaining the sealed arrangement of the container. As shown in fig. 6, after the device is actuated, the piercer pierces frangible portion 2713, thereby allowing reagent R to flow into reagent reservoir 2730 for subsequent use during the molecular diagnostic methods described herein. Specifically, as shown, the reagent plunger 2755 and piercer 2754 collectively move within the reagent container 2701 to pierce the frangible portion 2713 and push the reagent R toward the reagent reservoir 2730. Although reagent module 2700 is shown to include non-moving reagent container 2701 and moving piercer 2754, in other embodiments, the piercer may not be moving and the reagent container may be moving (see, e.g., reagent module 6700).

The detection module 2800 is configured to react the input solution S2 from the sample preparation module 2200 (or optionally the amplification module) with one or more reagents to generate a signal (or output) OP1 to indicate the presence or absence of a target organism in the biological sample S1. In particular, detection module 2800 defines a detection channel and includes a detection surface 2821 within the detection channel. The detection channel is (or can be placed in fluid communication with) each of sample preparation module 2200 and reagent module 2700. In this way, input solution S2 containing target amplicons may be transported into the detection channel and past detection surface 2821. Additionally, as shown in fig. 6, reagent R may also be transported into the detection channel and past detection surface 2821. The detection surface 2821 includes a series of capture probes to which target amplicons can bind as the input solution S2 flows across the detection surface 2821. The capture probe may be any suitable probe of the type described herein that is formulated to capture or bind the target amplicon. When reagent R reacts with captured input solution S2, a signal OP1 is generated from detection surface 2821.

The electronic control module 2950 is within the housing 2001 and may automatically control the heaters (e.g., the heaters 223), valves, pumps, power supplies, and/or any other components of the diagnostic device 2000 to facilitate molecular testing as described herein. The electronic control module 2950 may include a memory, a processor, an input/output module (or interface), and any other suitable module or software to perform the functions described herein. As shown in fig. 5 and 6, the electronic control module 2950 includes a switch 2906, which switch 2906, when actuated, initiates the molecular diagnostic test. The electronic control module 2950 may be powered by any suitable power source described herein, including the power source 1905 described above.

The cover 2050 is movably coupled to the housing 2001 and performs multiple functions, thereby facilitating actuation of the device 2000 with a single motion. As shown, lid 2050 includes a sealing portion 2053, a switching portion 2060, and a reagent actuator 2064. As indicated by arrow CC, the cover 2050 is configured to move relative to the housing 2001 from a first (or open) position (fig. 5) to a second (or closed) position (fig. 6). As shown in fig. 5, when the lid 2050 is in an open position, the seal 2053 (also referred to as a cover) is spaced from the input opening 2212. Similarly, when the lid 2050 is in the open position, the input opening 2212 is exposed, thereby allowing the biological sample S1 to be transferred into the sample preparation module 2200. After loading the biological sample S1, the user can close the lid 2050 (i.e., can move the lid to its second position). As shown in fig. 6, when the lid 2050 is in a closed position, the seal 2053 covers the input opening 2212. In some embodiments, the seal 2053 comprises a seal, gasket, or other material to fluidly isolate the sample input volume 2211 when the lid 2050 is in the second lid position.

In addition to covering the input opening 2212, closing the lid 2050 actuates other mechanisms within the device 2000. Specifically, as shown in fig. 6, when the lid 2050 is moved from an open position to a closed position, the switch portion 2060 actuates the switch 2906 to provide power to the electronic control module 2950 and/or the heater 2230. Additionally, when the lid 2050 is moved from an open position to a closed position, the reagent actuator 2064 releases reagent R from the sealed reagent container 2701. Specifically, as shown in fig. 6, reagent actuator 2064 applies a force to reagent plunger 2755, thereby moving reagent plunger 2755 and puncturer 2754. As shown in fig. 6, the piercer pierces frangible portion 2713, thereby allowing reagent R to flow into reagent reservoir 2730.

The molecular diagnostic test device 2000 (as well as any of the molecular diagnostic test devices described herein) may perform any of the "one-touch" actuation methods described herein. For example, FIG. 7 is a flow chart of a method 20 of detecting nucleic acids according to one embodiment. Although the method 20 is described as being performed on the apparatus 2000, in other embodiments, the method 20 may be performed on any suitable apparatus, such as the apparatus 6000 described below. The method 20 includes coupling a molecular diagnostic test device to a power source, at 22. The power source (not shown in fig. 5 and 6) may be any suitable power source, such as an alternating current (a/C) power source, a direct current (D/C) power source (e.g., a battery), a fuel cell, or the like.

At 23, the biological sample is transferred through the input opening into a sample preparation module within the molecular diagnostic test device. The biological sample S1 may be transferred into the device by any suitable mechanism, such as the sample transfer device 1110 described above. Biological sample S1 may be any suitable sample, such as, for example, blood, urine, a male urethral specimen, a vaginal specimen, a cervical swab specimen, a nasal swab specimen, a pharyngeal swab specimen, a rectal swab specimen, or any other biological sample described herein. Thus, in some embodiments, the biological sample S1 may be a "raw" (or unprocessed) sample.

The molecular diagnostic test device is then actuated at 24 by a single action of closing the lid to cover the input opening. The single action of the closure device causes the molecular diagnostic test device to perform a series of operations without any further user input. Referring to fig. 6, the lid 2050 may be closed by rotating the lid relative to the housing 2001. In other embodiments, the lid 2050 can be closed by a sliding action (see, e.g., device 6000), depressing a portion of the lid, or any other suitable closing mechanism. The molecular diagnostic test device may perform any of the methods described herein after being actuated through the cover opening. In particular, the act of closing the lid may also actuate an electronic control module (e.g., electronic control module 2950), release one or more reagents for use in a test (e.g., release reagent R into reagent reservoir 2730), and/or actuate any other mechanism within the device to facilitate the molecular diagnostic methods described herein. Specifically, at 24A, the device can heat the biological sample via the heater of the sample preparation module to lyse a portion of the biological sample, thereby generating an input sample. Referring to fig. 6, biological sample S1 may be heated by heater 2230 and the resulting lysed sample (i.e., input sample S2) may be transferred to detection module 2800 or an amplification module (not shown in fig. 6). In some embodiments, the method 20 optionally includes, at 24B, transferring the input sample to an amplification module within the molecular diagnostic test device. The input sample may then be heated within the reaction volume to amplify the nucleic acids within the input sample to produce an output solution comprising the target amplicons at operation 24C. The input solution may be amplified by using any suitable technique as described herein (e.g., PCR, isothermal amplification, etc.).

After amplification, the device then reacts, at 24D, within a detection module within the molecular diagnostic test device, each of the following: (i) an output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution. As shown in fig. 6, detection module 2800 includes a detection surface 2821 configured to capture target amplicons to generate an output signal OP 1. The output signal OP1 may be any suitable signal. In some embodiments, the output signal OP1 may be a colorimetric signal indicative of the presence of bound amplicons: if the target pathogen, target amplicon, and/or target organism is present, a colored product is formed, and if the target pathogen, target amplicon, and/or target organism is not present, a colored product is not formed.

The method further includes reading the result associated with the signal, at 25. In some embodiments, the readout can include visual inspection of the device and detection surface 2821 for colorimetric signals. In other embodiments, the signal OP1 generated by the detection surface 2821 is not necessarily visible to the naked eye. For example, in some embodiments, the readout may include scanning or otherwise receiving the signal OP1 using an auxiliary device, such as a mobile computing device. In still other embodiments, the reading of the results may include indirectly reading a secondary signal that conveys a result related to (or descriptive of) the primary output from detection surface 2821.

In some embodiments, method 20 optionally comprises discarding the molecular testing device after the readout. In some embodiments, the amount of sample and reagents may be such that the device can be disposed of by standard, unconstrained waste treatment procedures. In other embodiments, the discarding comprises disposing of the used device through standard medical waste disposal procedures. In some embodiments, method 20 optionally comprises storing the molecular diagnostic test device including any reagents sealed therein for at least 6 months prior to use.

In some embodiments, the molecular diagnostic test devices and related methods involve the use of multi-purpose reagents to perform both surface blocking and washing functions. In this way, the amount of reagent and the ease of use of the device may be increased, thereby facilitating bedside use, disposability of the device, and/or handling of the device in accordance with CLIA-exempt methods. In particular, in some embodiments, the multi-purpose reagent may include a blocking agent to reduce background signals associated with the attachment of undesirable particles during a detection event. By improving signal quality, such devices and methods may be adapted for use with limited sample preparation. In addition, the multi-purpose reagent may include a detergent that removes unbound components from within the detection module. Such methods may include delivering quantities of the multi-purpose agent at different times depending on the desired function of the agent.

Fig. 8-11 are schematic illustrations of a molecular diagnostic test device 3000 (also referred to as a "test device" or "device") including a multi-purpose reagent according to one embodiment. The testing device 3000 is configured to manipulate a biological sample to produce one or more output signals associated with a target cell according to any of the methods described herein. In some embodiments, the testing device 3000 may be an integrated device suitable for use within a bedside facility (e.g., a doctor's office, pharmacy, etc.), a distributed testing facility, or in the home of a user. Similarly, in some embodiments, the modules of the devices described below are housed within a single housing so that the testing device can operate entirely without any additional instrumentation, docking stations, or the like. Further, in some embodiments, the device 3000 may have a size, shape, and/or weight such that the device 3000 may be carried, held, used, and/or operated in a user's hand (i.e., it may be a "handheld" device). In some embodiments, the testing device 3000 may be a self-contained single-use device.

In some embodiments, the device 3000 (as well as any of the devices shown and described herein) may be a CLIA-exempt device and/or may operate according to a CLIA-exempt method. Similarly, in some embodiments, the device 3000 (as well as any of the other devices shown and described herein) is configured to operate in a sufficiently simple manner and can produce sufficiently accurate results, thereby creating a limited likelihood of misuse and/or a limited risk of harm when improperly used. In some embodiments, the device 3000 (as well as any of the other devices shown and described herein) may be operated by a user with little (or no) scientific training, in accordance with methods that require little user judgment and/or in which certain operational steps are easily controlled and/or automatically controlled. In some embodiments, the molecular diagnostic test device 3000 may be configured for long-term storage in a manner that creates a limited likelihood of improper use (reagent damage, reagent expiration, reagent leakage, etc.). In some embodiments, the molecular diagnostic test device 3000 is configured to be stored for up to about 36 months, up to about 32 months, up to about 28 months, up to about 24 months, up to about 20 months, up to about 18 months, up to 12 months, up to 6 months, or up to any time therebetween.

Testing device 3000 includes housing 3001, sample preparation module 3200 (also referred to as a sample staging module), reagent module 3700, and detection module 3800. In some embodiments, the testing device 3000 can include any other component or module described herein, such as, for example, an amplification module (e.g., amplification module 1600 or 6600), a rotary valve (e.g., to control the flow of reagents and/or samples, such as valve 6300), or a fluid transfer module (e.g., fluid transfer module 6400). Housing 3001 may be any structure in which sample preparation module 3200 or other components are housed (or partially housed) to form an integrated device for sample preparation and/or molecular testing.

Sample preparation module 3200 defines a sample input volume 3211 that receives biological sample S1. Sample preparation module 3200 may include any of the components as described herein to manipulate biological sample S1 for further diagnostic testing and/or to generate a solution to detect nucleic acids. For example, in some embodiments, sample preparation module 3200 may comprise one or more heaters, one or more chambers within which biological sample S1 may be manipulated, one or more mixing chambers, and/or certain on-board reagents (e.g., lysis buffer, RT enzymes, control organisms, etc.). In some embodiments, the sample preparation module 3200 is configured to extract nucleic acid molecules from the biological sample S1 and may generate an input solution S2 (see fig. 10), the input solution S2 optionally being transported into an amplification module (not shown), or into the detection module 3800.

The reagent module 3700 is disposed within the housing 3001 and includes a first reagent container 3701, a first reagent actuator 3755, a second reagent container 3702, and a second reagent actuator 3765. The reagent module 3700 provides on-board preservation of the first reagent R1 (within the first reagent container 3701) and the second reagent R2 (within the second reagent container 3702) used in connection with the molecular diagnostic tests described herein. In some embodiments, the first reagent R1 is sealed within the first reagent container 3701 and the second reagent R2 is sealed within the second reagent container 3702. In some embodiments, reagent module 3700 can include one or more puncturers (see, e.g., the puncturer of reagent module 2700 or the puncturer of reagent module 6700) that can release reagent for use upon actuation of the device.

The first reagent R1 is a multipurpose reagent and includes a blocking agent and a wash buffer. In some embodiments, the blocking agent comprises bovine serum albumin and the wash buffer comprises a detergent. Further, in some embodiments, the first reagent R1 includes 0.02% to 5% bovine serum albumin and 0.05% to 10% detergent. The inclusion of a blocking agent can help achieve reproducible and accurate results in methods like those described herein that employ limited sample preparation (i.e., limited filtration, separation, etc.). In particular, molecules that are undesirable for producing an output signal associated with a target nucleic acid (i.e., "undesired molecules") may be attached to a surface in detection module 3800 when biological sample S1 is subjected to limited sample preparation. The undesired attachment of molecules, especially on non-detection surfaces, can lead to the generation of undesirable background signals. By including a blocking agent, the first reagent R1 may be used to deliver a blocking agent into the detection module 3800 to limit the attachment of the undesired molecules. Similarly, the first reagent R1 may be used to apply a coating within the detection module to limit unwanted background signals, as described herein. In other embodiments, the blocking agent within the first reagent R1 may be casein, skim milk solids, gelatin, and the like. In yet other embodiments, the blocking agent within the first reagent R1 may be a non-biological blocking agent. Furthermore, by also including a detergent in the first reagent R1, the first reagent R1 may also be used (e.g., at a different time) to remove unbound components from the detection module 3800 during a detection event.

In some embodiments, the first reagent R1 may also include a humectant to increase the likelihood that the first reagent R1 will substantially coat surfaces within the detection module 3800. In some embodiments, the first reagent R1 may also include an antimicrobial component to improve the shelf life of the device 3000.

The second reagent R2 may be a detection reagent formulated to facilitate generation of a signal indicative of the presence of the target amplicon from the input solution S2. In some embodiments, the second reagent R2 can be formulated to include a binding moiety and any suitable enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase. In some embodiments, the HRP enzyme has been conjugated to a streptavidin molecule. In some embodiments, the second reagent R2 can be a substrate that when catalyzed generates a colored molecule.

The detection module 3800 is configured to react the input solution S2 from the sample preparation module 3200 (or optionally an amplification module) with a second reagent R2 to generate one or more signals (or outputs) OP1, OP2, indicating the presence or absence of a target organism in the biological sample S1. In particular, the detection module 3800 defines a detection channel and includes a first detection surface 3821 and a second detection surface 3822 within the detection channel. The detection module 3800 also includes a non-detection surface 3826, the non-detection surface 3826 abutting, wrapping around, or contacting either or both of the first detection surface 3821 and the second detection surface 3822. As described above, by limiting any background signal generated by the non-detection surface 3826, the overall accuracy of the apparatus 3000 and related molecular diagnostic methods may be improved.

The detection channel is in fluid communication with (or can be placed in fluid communication with) each of sample preparation module 3200 and reagent module 3700. In this way, the input solution S2 containing the target amplicons can be transported into the detection channel and past the detection surface 3821. In addition, as shown in FIG. 11, a second reagent R2 may also be delivered into the detection channel and past the detection surface 3821,3822. The detection surface 3821,3822 includes a series of capture probes to which target amplicons can bind as the input solution S2 flows across the detection surface 3821,3822. The capture probe may be any suitable probe of the type described herein that is formulated to capture or bind the target amplicon. When the second reagent R2 reacts with the captured input solution S2, a first signal OP1 is generated from the first detection surface 3821 and a second signal OP2 is generated from the second detection surface 3822.

The molecular diagnostic test device 3000 (as well as any of the molecular diagnostic test devices described herein) can perform any of the methods described herein. For example, FIG. 12 is a flow chart of a method 30 of detecting nucleic acids according to one embodiment. Although the method 30 is described as being performed on the apparatus 3000, in other embodiments, the method 30 may be performed on any suitable apparatus, such as the apparatus 6000 described below. The method 30 optionally includes storing the molecular diagnostic test device including any reagents sealed therein for at least 6 months prior to use, at 32. For example, the device 3000 including the first reagent R1 and the second reagent R2 may be stored as part of a holding plan for at least 6 months.

To initiate a molecular diagnostic test, the method 30 optionally includes transferring the biological sample to a sample preparation module within the molecular diagnostic test device. Referring to fig. 8, the biological sample S1 may be transferred into the device by any suitable mechanism, such as a sample transfer device 3110. Biological sample S1 may be any suitable sample, such as, for example, blood, urine, a male urethral specimen, a vaginal specimen, a cervical swab specimen, a nasal swab specimen, a pharyngeal swab specimen, a rectal swab specimen, or any other biological sample described herein. Thus, in some embodiments, the biological sample S1 may be a "raw" (or unprocessed) sample.

At 33, a first volume of a first reagent R1 is transferred at a first time from a reagent module within the molecular diagnostic test device to a detection module within the molecular diagnostic test device. The detection module may be similar to detection module 3800 and includes a detection surface 3821 and one or more non-detection surfaces 3826 configured to capture target amplicons associated with the nucleic acids. As described above, the first volume of the first reagent R1 contains an amount of the blocking solution sufficient to adsorb to surfaces within the detection module 3800 (including the detection surface 3821 and the non-detection surface 3826). Referring to fig. 9, the first volume can be transferred by moving the first agent actuator 3755 as indicated by arrow DD. The flow of the first volume of the first reagent R1 is shown by arrow EE. In some embodiments, the first time (i.e., the time the first portion is passed into the detection module) is sufficient before the remaining operations of the method allow the blocking agent to be sufficiently coated and adsorbed within the detection module 3800. For example, in some embodiments, the first time lasts at least 3 minutes before a subsequent step involving flowing a solution into the detection module. In some embodiments, for example, a first volume of the first reagent R1 may be transferred into the detection module 3800 while the biological sample S1 is heated and/or processed in the sample input module 3200. In this way, the "seal operation" is not added to the total test duration.

The biological sample S1 may be heated within the sample preparation module 3200, and the resulting lysed sample (i.e., input sample S2) may be transported towards the amplification module 3800 or the amplification module (not shown in fig. 8-11). In some embodiments, the method 30 optionally includes, at 34, transferring the input sample to an amplification module within the molecular diagnostic test device. At 35, the input sample may then be heated within the reaction volume to amplify the nucleic acids within the input sample, thereby producing an output solution comprising the target amplicons. The input solution can be amplified by using any suitable technique (e.g., PCR, isothermal amplification, etc.), as described herein.

After optional amplification, the method includes, at 36, transferring a sample solution comprising target amplicons into the detection module at a second time such that the target amplicons are captured on the detection surface. Referring to fig. 10, the sample (or input) solution is represented by arrow S2. As described above, the first detection surface 3821 and the second detection surface 3822 each include a series of capture probes to which target amplicons can bind as the input solution S2 flows across the detection surface 3821,3822. Furthermore, by applying a blocking agent to the surface within the detection module, the possibility of non-specific protein adsorption is reduced. In some embodiments, the method may optionally include delivering an amount of the first reagent R1 to a detection module, thereby washing unbound components from the detection module. In particular, the first reagent solution comprises a wash buffer in an amount sufficient to remove unbound components from at least one of the sample solution or the second reagent solution from the detection module. As shown in fig. 10, the first reagent R1 may be transferred into the detection module by further actuating the first reagent actuator 3755.

Referring to FIG. 12, at 37, after a second time, a second reagent is delivered to the detection module. As shown in fig. 11, the second reagent R2 may be delivered by moving the second reagent actuator 3765 as indicated by arrow FF. As shown, the second reagent R2 may flow across the detection surface. The second reagent may be reagent R2 described above and is formulated to cause generation of a signal indicative of the presence of the target amplicon within the sample solution. The method further includes, at 38, transferring a second volume of the first reagent to the detection module after a second time. The second volume of the first reagent solution comprises a wash buffer in an amount sufficient to remove unbound components from at least one of the sample solution or the second reagent solution from the detection module.

In some embodiments, the method optionally includes delivering a third reagent to the detection module at 39. The third reagent may be, for example, a substrate or other substance formulated to produce a signal when catalyzed by the second reagent R2. In this way, the device can react each of (i) the output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution. In some embodiments, the method comprises providing a continuous flow of the third reagent through the detection module. In particular, in some embodiments, the third reagent comprises a precipitating substrate formulated to produce a coloured molecule when catalysed by the second reagent R2 captured on the detection surface. Since the third reagent is a precipitation substrate, the generated coloured molecules will settle on the detection surface. Furthermore, by continuously replenishing the third reagent (i.e., precipitating the substrate), the reaction that produces the colored molecule will not be limited by the concentration (or amount) of the third reagent. Similarly, by flowing the third reagent continuously over the detection surface (and the captured second reagent R2), the reaction that produces the coloured molecule will be diffusion limited. Instead, the reaction will be kinetically (or rate) limited and therefore will be faster than maintaining a set amount of the third reagent in the detection module.

In some embodiments, the method optionally comprises reading the results associated with the signal, as described herein.

Although fig. 9 and 10 illustrate the first volume of the first reagent R1 and the second volume of the first reagent R1 being transferred by moving the first reagent actuator 3755 in the same direction, in other embodiments, any suitable mechanism may be employed for transferring the desired amount of the first reagent R1. For example, in some embodiments, the first reagent may be recycled (or reused) within the apparatus 3000 (or any other apparatus). In particular, a first volume of a first reagent may be applied for containment purposes and then may be returned to the reagent module for subsequent reuse for washing purposes. By recycling the first reagent for multiple purposes, the amount of reagent required is reduced, which allows for smaller packaging, lower cost, and the like.

For example, fig. 13 is a flow chart of a method 40 of detecting nucleic acids according to one embodiment. Although the method 40 is described as being performed on the apparatus 3000, in other embodiments, the method 40 may be performed on any suitable apparatus, such as the apparatus 6000 described below. To initiate the molecular diagnostic test, the method 40 includes, at 42, transferring the biological sample to a sample preparation module within the molecular diagnostic test device. Biological sample S1 may be any suitable sample, such as, for example, blood, urine, a male urethral specimen, a vaginal specimen, a cervical swab specimen, a nasal swab specimen, or any other biological sample described herein. Thus, in some embodiments, the biological sample S1 may be a "raw" (or unprocessed) sample.

At 43, the molecular diagnostic test device is then actuated (e.g., in some embodiments, actuated by a single motion) causing the molecular diagnostic test device to perform a series of operations. As a result of the actuation, a first volume of the first reagent R1 is transferred from the reagent module within the molecular diagnostic test device to the detection module within the molecular diagnostic test device at a first time at 43A. The detection module may be similar to detection module 3800 and includes a detection surface 3821 and one or more non-detection surfaces 3826 configured to capture target amplicons associated with the nucleic acids. As described above, the first volume of the first reagent R1 contains an amount of blocking solution sufficient to adsorb to surfaces within the detection module 3800 (including the detection surface 3821 and the non-detection surface 3826). At 43B, the device then transfers the first volume of the first reagent R1 back to the reagent module. This may be accomplished, for example, by moving the first agent actuator 3755 in a direction opposite to that indicated by arrow DD in figure 9 to aspirate the first agent R1 back into the agent module 3700. In some embodiments, the method can include allowing a first volume of the first reagent to remain in the detection module for a residence time to allow a blocking function (i.e., adsorption) to occur. The residence time may be, for example, at least 1 minute, 2 minutes, at least 3 minutes, or at least 4 minutes. In some embodiments, the method can include heating the detection module (e.g., to a temperature of at least 30C, 40C, or 50C) to promote adsorption.

At operation 43C, the device can heat the biological sample with a heater to produce an output sample comprising the target amplicons. In other words, the input sample can be heated within the reaction volume to amplify the nucleic acids within the input sample, thereby producing an output solution comprising the target amplicons. The input solution can be amplified by using any suitable technique (e.g., PCR, isothermal amplification, etc.), as described herein.

After amplification, the method includes delivering a sample solution comprising a target amplicon to a detection module at a second time such that the target amplicon is captured on a detection surface at 43D. At 43E, the device then transfers the second volume of the first reagent into the detection module. The second volume of the first reagent solution comprises a wash buffer in an amount sufficient to remove unbound components from at least one of the sample solution or the second reagent solution from the detection module.

The method further includes reading a result associated with the signal, at 44. In some embodiments, the readout may include visual inspection of the device and the detection surface 3821,3822 for a colorimetric signal. In other embodiments, the signal OP1 generated by the detection surface is not necessarily visible to the naked eye. For example, in some embodiments, the readout may include scanning or otherwise receiving the signals OP1, OP2 using an auxiliary device, such as a mobile computing device. In still other embodiments, the reading can include indirectly reading a secondary signal that conveys a result related to (or descriptive of) the primary output from the detection surface.

Fig. 14 illustrates a portion of operations and/or features related to an enzyme reaction, which may be performed by or within detection module 3800, detection module 4800, or any other detection module described herein (e.g., detection module 6800), according to an embodiment. In some embodiments, the enzymatic reaction may be performed to facilitate visual detection of molecular diagnostic test results using device 3000, device 4000, device 5000, device 6000, or any other device or system described herein. In other embodiments, the enzymatic reaction need not be performed to produce a visual inspection. For example, as described herein, in some embodiments, methods employing the illustrated enzymatic reactions may employ alternative methods to read results related to the generated signal.

In some embodiments, the reaction, detection module 4800 and/or the remaining components within the device 4000 (or device 6000) can be collectively configured such that the device is a single-use device that can be used at a bedside facility and/or at a user's home. Similarly, in some embodiments, the apparatus 4000 (as well as any of the other apparatuses shown and described herein) may be configured for use in a decentralized testing facility. Moreover, in some embodiments, the reaction shown in fig. 14 can facilitate device 4000 (as well as any of the other devices shown and described herein) to operate with sufficient ease and accuracy to be a CLIA-exempt device. Similarly, in some embodiments, the reaction shown in fig. 14 may provide output signal OP1 in a manner that creates a limited likelihood of improper use and/or a limited risk of harm when improperly used. In some embodiments, the reaction can be successfully completed within the device 4000 (or any other device described herein) after actuation by a user with little (or no) scientific training, in accordance with methods that require little user judgment and/or in which certain operational steps are easily controlled and/or automatically controlled.

As shown, detection module 4800 includes a detection surface 4821 within a read lane or flow channel. The detection surface 4821 is spotted and/or covalently bonded to a specific hybridization probe 4870, such as an oligonucleotide. Hybridization probes 4870 (also referred to as capture probes) can be similar to any of the capture probes described herein, including those described in connection with detection surface 3821. In some embodiments, hybridization probe 4870 is specific for a target organism, nucleic acid, and/or amplicon. Bonding of hybridization probes 4870 to detection surface 4821 can be performed using any suitable procedure or mechanism. For example, in some embodiments, hybridization probe 4870 can be covalently bonded to detection surface 4821.

Reference numeral S3 illustrates biotinylated amplicons that result from an amplification step, such as, for example, by amplification module 4600 of fig. 15 (or any other amplification module described herein). Biotin can be incorporated in any suitable manner within the amplification procedure and/or within the amplification module 4600. As indicated by arrow XX, the output from the amplification module, including biotinylated amplicon S3, travels in the read long lane and passes through detection surface 4821. Hybridization probe 4870 is formulated to hybridize to target amplicon S3 present in a flow channel and/or near detection surface 4821. The detection module 4800 and/or detection surface 4821 is heated to incubate biotinylated amplicon S3 in the read long lane for several minutes in the presence of hybridization probe 4870, allowing binding to occur. In this way, target amplicon S3 is captured and/or immobilized on detection surface 4821, as shown. Although disclosed as being labeled with biotin, in other embodiments, the target molecule may be labeled in any suitable manner that will allow binding of the complex comprising the sample molecule binding moiety and an enzyme capable of facilitating a colorimetric reaction. For example, in some embodiments, the target molecule may be labeled with one or more of the following: streptavidin, fluorescein, texas red, digoxin or fucose.

In some embodiments, a first wash solution (not shown in fig. 14) can be conveyed past the detection surface 4821 and/or within the flow channel to remove unbound PCR products and/or any residual solution. Such wash solutions may be, for example, multi-purpose reagents, as described above with reference to apparatus 3000 and first reagent R1 of methods 30 and 40. However, in other embodiments, no washing operation is performed.

The detection reagent R5 travels in the read long lane and passes over the detection surface 4821 as indicated by arrow YY. The detection reagent R5 may be any of the detection reagents described herein. In some embodiments, the detection reagent R5 can be a horseradish peroxidase (HRP) enzyme ("enzyme") with a streptavidin linker. In some embodiments, streptavidin and HRP are cross-linked to provide bifunctional. As shown, the detection reagent binds to the captured amplicon S3. Detection module 4800 and/or detection surface 4821 is heated to incubate detection reagent R5 in the presence of biotinylated amplicon S3 for several minutes in the read long lane to facilitate binding.

In some embodiments, a second wash solution (not shown in fig. 14) may be delivered across the detection surface 4821 and/or within the flow channel to remove unbound detection reagent R5. Such wash solutions may be, for example, multi-purpose reagents, as described above with reference to apparatus 3000 and first reagent R1 of methods 30 and 40. However, in other embodiments, the second washing operation is not performed.

The detection reagent R6 travels in the read long lane and passes over the detection surface 4821 as indicated by arrow ZZ. The detection reagent R6 may be any of the detection reagents described herein. In some embodiments, the detector reagent R6 can be, for example, a substrate formulated to enhance, catalyze, and/or promote the production of the signal OP1 when reacted with the detector reagent R5. Specifically, the substrate is formulated such that upon contact with the detection reagent R5 (HRP/streptavidin) a colored molecule is produced. Thus, a colorimetric output signal OP1 is formed where HRP attaches to the amplicon. The color of the output signal OP1 indicates the presence of bound amplicons: if the target pathogen, target amplicon, and/or target organism is present, a colored product is formed, and if the target pathogen, target amplicon, and/or target organism is not present, a colored product is not formed.

As described above with respect to method 30, in some embodiments, the detection reagent R6 can flow continuously across the detection surface 4821 to ensure that the reaction that produces the chromonic molecule is not limited by the availability of detection reagent. Further, in some embodiments, the detection reagent R6 can be a precipitation substrate.

In some embodiments, the methods comprise lysing the original sample and performing reverse transcription Polymerase Chain Reaction (PCR) on the lysed sample to facilitate detection of the target RNA, e.g., for detection of the target virus. To facilitate such methods, in some embodiments, the device may include a reverse transcription module to facilitate such methods of isolating and detecting viruses. As an example, fig. 15 is a schematic illustration of a molecular diagnostic test device 4000 (also referred to as a "test device" or "device") including a reverse transcription module 4270, according to one embodiment. The schematic illustration depicts the main components of the testing apparatus 4000.

The testing device 4000 is an integrated device (i.e., the modules are housed within a single housing) that is suitable for use within a bedside facility (e.g., a doctor's office, pharmacy, etc.), a distributed testing facility, or within the home of a user. In some embodiments, the device 4000 may have a size, shape, and/or weight such that the device 4000 may be carried, held, used, and/or operated in a user's hand (i.e., it may be a "handheld" device). The handheld device may have a size of less than 15cmx15cmx15cm, or less than 15cmx15cmx10cm, or less than 12cmx12cmx6 cm. In other embodiments, the test device 4000 may be a self-contained single-use device. Similarly, the test device 4000 is a self-contained device that includes all necessary substances, mechanisms, and components to perform any of the molecular diagnostic tests described herein. As such, the apparatus 4000 does not require any external instruments to manipulate the biological sample, and only needs to be connected to a power source (e.g., to an a/C power source, coupled to a battery, etc.) to accomplish the methods described herein. In some embodiments, the testing device 4000 may be configured with a lock or other mechanism to prevent or attempt to reuse the device.

Further, in some embodiments, device 4000 may be a CLIA-exempt device and/or may operate according to a CLIA-exempt method. Similarly, in some embodiments, device 4000 (as well as any of the other devices shown and described herein) is configured to operate in a sufficiently simple manner, and can produce sufficiently accurate results to create a limited likelihood of misuse and/or a limited risk of harm when improperly used. In some embodiments, the device 4000 (as well as any of the other devices shown and described herein) can be operated by a user with little (or no) scientific training, in accordance with methods that require little user judgment and/or in which certain operational steps are easily controlled and/or automatically controlled. In some embodiments, the molecular diagnostic test device 4000 may be configured for long-term storage in a manner that creates a limited likelihood of improper use (reagent damage, reagent expiration, reagent leakage, etc.). In some embodiments, the molecular diagnostic test device 4000 is configured to be stored for up to about 36 months, up to about 32 months, up to about 26 months, up to about 24 months, up to about 20 months, up to about 48 months, or up to any value therebetween.

Testing device 4000 is configured to operate biological sample S1 to generate one or more output signals associated with the target cells. Specifically, the apparatus 4000 includes an actuator 4050, a sample preparation (or staging) module 4200, a fluid drive (or fluid transfer) module 4400, a mixing module 4250, an amplification module 4600, a detection module 4800, a reagent module 4700, a valve 4300, and a power and control module (not shown). The testing device and certain components thereof may be similar to many of the components of the device 6000 shown and described with reference to fig. 19. Accordingly, the actuator 4050, the fluid drive (or fluid transfer) module 4400, the mixing module 4250, the amplification module 4600, the detection module 4800, the reagent module 4700, and the valve 4300 are not described in detail herein. Further, the device comprising a reverse transcription module is similar to the reverse transcription device shown and described in international patent publication No. WO2018/005870 entitled "device and method for nucleic acid extraction", the entire contents of each of which are incorporated herein by reference.

Apparatus 4000 differs from apparatus 1000, apparatus 2000, apparatus 3000 and apparatus 6000 in that sample preparation module 4200 includes lysis chamber 4201 and reverse transcription module 4270. Lysis chamber 4201 can be similar to the lysis chamber shown and described in international patent publication No. WO2018/005710, entitled "apparatus and method for detecting molecules using a flow cell," which is incorporated herein by reference in its entirety. Specifically, the lysis module 4300 includes a chamber body and a heater. In use, a sample (filtered sample or raw biological sample S1) is transferred into the chamber and can be heated to a first temperature within the lysis temperature range to release ribonucleic acid (RNA) molecules. The heater may deliver thermal energy to the lysis module 4300 to produce a lysis temperature zone within any desired portion of the lysis module 4300 for any time period described herein. Thus, the lysis module can lyse cells within a biological sample, as well as target viruses that may reside within the cells to produce RNA suitable for use in a reverse transcription process.

After completion of lysis, the lysed sample may then be mixed with a reverse transcriptase to form a reverse transcription solution. The mixing may be performed in any suitable part of the apparatus, such as, for example, in the flow path between lysis module 4201 and reverse transcription module 4270. Alternatively, in some embodiments, mixing of the lysed sample with the reverse transcriptase may be performed within mixing module 4250.

The reverse transcription module 4270 is integrated in the device and includes a flow member and a heater. The flow member defines a reverse transcription flow path through which a lysed sample comprising the RNA can be transported. The reverse transcription module 4270 is configured to heat the reverse transcription solution to a second temperature within the reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules. In some embodiments, the reverse transcription module 4270 is configured to heat the reverse transcription solution to a third temperature above the inactivation temperature to cause inactivation of the reverse transcriptase. The reverse transcription solution may then be transferred to the mixing module 4250 and mixed with PCR reagents. After mixing, the solution may then be transferred to the amplification module 4600 and amplified in the manner described herein.

Although apparatus 4000 is shown and described as including a lysis module 4300 separate from a reverse transcription module 4270, in other embodiments, the apparatus and molecular diagnostic methods may include a single chamber or module within which a) a sample may be lysed to produce RNA, B) the RNA may be heated to produce complementary deoxyribonucleic acid (cDNA), and C) the solution may be further heated to inactivate reverse transcriptase (i.e., RT enzyme). Similarly, in some embodiments, the method comprises lysing the original sample and performing a reverse transcription Polymerase Chain Reaction (PCR) on the lysed sample in the same environment. In other words, in some embodiments, the device includes a single lysis/RT-PCR module to facilitate methods that include lysing the original sample in a single chamber and performing rapid RT-PCR. Such methods can be performed in a manner that limits degradation of the target RNA after cleavage, thereby producing accurate results. Thus, such methods are suitable for execution by CLIA-exempt bedside devices.

Fig. 16 is a schematic illustration of a portion of a molecular diagnostic test device 5000 (also referred to as a "test device" or "device"), the molecular diagnostic test device 5000 including a sample preparation (or fractionation) module 5200 that can perform lysis, RT-PCR, enzyme inactivation in a single environment (or module). The testing device 5000 may have similar features to the device 4000 described above, and be an integrated device (i.e., the modules are housed in a single housing) suitable for use in a bedside facility (e.g., a doctor's office, pharmacy, etc.), a distributed testing facility, or in the home of the user. The sample preparation module 5200 includes input (or housing) reservoirs 5211 and flow channels 5214 inside which input sample S1 can be heated to perform RT-PCR, among other methods. The sample preparation module further comprises a reverse transcriptase R2, said reverse transcriptase R2 being mixed with the biological sample S1. Upon completion of the RT-PCR process, the solution is then transferred to a mixing module 5250 where it is mixed with amplification reagents R3 suitable for performing the desired amplification (e.g., PCR or other amplification method). All or a portion of device 5000 may be included in any of the devices described herein. Furthermore, the apparatus 5000 may be used to perform any of the RT-PCR methods described herein.

Fig. 17A shows a graph of temperature as a function of time, and fig. 18 is a flow chart of a method 50 of performing lysis, reverse transcription, and inactivation processes in a single module within a handheld single use device. Although method 50 is described in conjunction with the temperature performance graph of fig. 17A, apparatus 5000, and apparatus 6000 (described below), in other embodiments, RT-PCR method 50 may be performed using any suitable apparatus as described herein. At 52, method 50 includes mixing a reverse transcriptase enzyme with a biological sample within a sample preparation module to form a reverse transcription solution. The sample preparation module can be a single environment or module, such as sample preparation module 6200 described below. In some embodiments, the reverse transcriptase can be reagent R4 in lyophilized or solid form, which is captively held in a retention volume or mixing volume (e.g., retention volume 6211) of the sample preparation module. In some embodiments, the biological sample may be a raw and/or unfiltered sample. In some embodiments, the reverse transcription solution may be free of ribonuclease inhibitors. Specifically, as described herein, in some embodiments, method 50 may be performed in the following manner: the released RNA is rapidly subjected to reverse transcription so that degradation of RNA by ribonuclease is limited.

The reverse transcription solution is then heated within the sample preparation module to a first temperature within the lysis temperature range to release ribonucleic acid (RNA) molecules at 53. The cracking temperature range may be any of the ranges described herein. For example, in some embodiments, the first temperature range may be from about 25C to about 40C. In some embodiments, heating can be performed by a segmented or "multizone" heater (e.g., heater 6230) that transfers thermal energy into the initial volume 6211 of the sample preparation module. Referring to fig. 17A, in some embodiments, heating to the first temperature may include heating the reverse transcription solution at a ramp rate (as shown in region 61 in fig. 17A). In other words, in some embodiments, the reverse transcription solution mayAccording to the temperature from the initial temperature to the reverse transcription temperature T_rtAnd is not necessarily maintained at a constant cracking temperature for a set period of time. In this way, the solution can be passed through a lysis temperature range (e.g., 25C-35C) while being heated toward the target reverse transcription temperature. However, in other embodiments, the method 50 may include maintaining the reverse transcription solution at a constant lysis temperature for a set period of time.

At 54, the reverse transcription solution is then heated within the sample preparation module to a second temperature within the reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules from the released RNA. The reverse transcription temperature range may be any of the ranges described herein. For example, in some embodiments, the first temperature range may be from about 40C to about 60C. In some embodiments, heating can be performed by a segmented or "multizone" heater (e.g., heater 6230) that transfers thermal energy into the initial volume 6211 of the sample preparation module. In other embodiments, the reverse transcription solution can be delivered through a serpentine flow channel (e.g., channel 6214) to facilitate heating by heater 6230. Referring to fig. 17, in some embodiments, heating to the second temperature may include heating the reverse transcription solution and then maintaining the solution at a substantially constant target reverse transcription temperature T_rtFor a period between t1 and t2, as shown by region 62 in fig. 17. However, in other embodiments, the reverse transcription solution may be continuously heated such that the temperature is in the direction of the inactivation temperature T_inactAnd is not necessarily maintained at a constant reverse transcription temperature for a set period of time.

In some embodiments, the solution may be maintained at the second temperature (e.g., Trt) for a suitable period of time (e.g., see FIG. 17A, between the first time (t1) and the second time (t2) to complete the reverse transcription reaction, hi some embodiments, the time may be about 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, and at least 5 minutes.

The method further includes heating the reverse transcription solution to a third temperature above the inactivation temperature within the sample preparation module to cause inactivation of the reverse transcriptase, at 55. The inactivation temperature range may be any of the ranges described herein. For example, in some embodiments, the first temperature range may be greater than about 92C, 93C, 94C, 95C, 96C, 97C, 98C, and about 99C. In other embodiments, the RT enzyme may be inactivated at a much lower temperature, and the first temperature range may be greater than about 56C, 58C, 60C, 62C, 64C, 68C, 75C, and about 80C. In some embodiments, the third temperature may be maintained for a suitable period of time (see fig. 17A, from time t3 to time t4, providing a suitable amount of time to inactivate the RT enzyme). In some embodiments, heating can be performed by a segmented or "multizone" heater (e.g., heater 6230) that transfers thermal energy into the initial volume 6211 of the sample preparation module. In other embodiments, the reverse transcription solution can be delivered through a serpentine flow channel (e.g., channel 6214) to facilitate heating by heater 6230.

The reverse transcription solution is then transferred to an amplification module at 56. Any additional nucleic acid detection methods, such as further amplification of cDNA, can be accomplished according to the methods described herein.

Although fig. 17A shows lysis and RT-PCR as being performed in different steps, in some embodiments, the method may include performing these operations in a sequential manner. Similarly, in some embodiments, the method may comprise lysing the cells and/or virus to release RNA and producing cDNA from the released RNA in a continuous, substantially simultaneous operation. In this way, the time between the release of RNA and the transcription process to produce cDNA can be minimized, such that the potential degradation of RNA by endogenous ribonucleases is limited. This further allows any of the methods described herein to be accomplished without the use of ribonuclease inhibitors or other RNA protection mechanisms (e.g., bead capture, additional filtration, etc.). This method has advantageously been found to be effective for certain viruses, including MS phages and influenza a viruses. In other embodiments, this sequential lysis/RT-PCR method can be performed in a detection assay for HIV and all Hantavirus (Hantavirus) species.

Fig. 17B shows a temperature/time performance graph of a method according to one embodiment. The RT-PCR method may be performed using any suitable apparatus as described herein, and may include mixing a reverse transcriptase with a biological sample within a sample preparation module to form a reverse transcription solution. The sample preparation module can be a single environment or module, such as sample preparation module 6200, described below. In some embodiments, the reverse transcriptase can be reagent R4 in lyophilized or solid form, which is captively held in a retention volume or mixing volume (e.g., retention volume 6211) of the sample preparation module. In some embodiments, the biological sample may be a raw and/or unfiltered sample. In some embodiments, the reverse transcription solution may be free of ribonuclease inhibitors. Specifically, as described herein, in some embodiments, the method may be performed in the following manner: the released RNA is rapidly subjected to reverse transcription so that degradation of RNA by ribonuclease is limited.

The reverse transcription solution is then heated within the reaction volume of the sample preparation module to a first temperature within the lysis temperature range to release ribonucleic acid (RNA) molecules. The cracking temperature range may be any of the ranges described herein. For example, in some embodiments, the first temperature range may be from about 25C to about 40C. Referring to fig. 17B, in some embodiments, heating to the first temperature may include heating the reverse transcription solution at a ramp rate, as shown in region 71. In other words, in some embodiments, the reverse transcription solution may be at a temperature from the initial temperature to the reverse transcription temperature T_RTAnd is not necessarily maintained at a constant cracking temperature for a set period of time. In this way, the solution can pass through a cleavage temperature range (e.g., 25C-35C) and/or a particular cleavage temperature T_Cracking(T_LYSIS) While being heated toward the target reverse transcription temperature.

The reverse transcription solution is then heated within the reaction volume to a second temperature within the reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules from the released RNA. The reverse transcription temperature range may be any of the ranges described herein. For example, in some embodiments, the first temperature range may be from about 40C to about 60C. In some embodiments, heating can be performed by a segmented or "multizone" heater (e.g., heater 6230) that transfers thermal energy into the initial volume 6211 of the sample preparation module. In other embodiments, the reverse transcription solution can be delivered through a serpentine flow channel (e.g., channel 6214) to facilitate heating by heater 6230. Referring to fig. 17B, in some embodiments, the reverse transcription solution may be continuously heated such that the temperature increases along a second ramp rate toward and/or through the reverse transcription temperature, as shown in region 72, and is not necessarily held at a constant reverse transcription temperature for a set period of time.

In some embodiments, the heating to the first temperature and the heating to the second temperature are performed continuously such that cDNA is produced in less than 1 minute of releasing the RNA molecule. In some embodiments, the heating to the first temperature and the heating to the second temperature are performed continuously such that cDNA is produced in less than 30 seconds of releasing the RNA molecule.

In some embodiments, the solution can then be passed to a mixing module (e.g., mixing assembly 6250) where the DNA polymerase is mixed into the solution. This is shown by region 73 in fig. 17B. In some embodiments, the solution may then be transferred to an amplification module (e.g., amplification module 6600) where the solution may be further heated to a) activate DNA polymerase and B) inactivate RT enzyme. This is shown by area 74 in fig. 17B. The solution may then be subjected to thermal cycling according to the methods described herein, as shown by region 75 in fig. 17B.

In some embodiments, heating to a first temperature (for lysis) and heating to a second temperature (for RT-PCR) may be performed at different ramp rates, as shown in fig. 17C.

Fig. 19 is a schematic illustration of a molecular diagnostic test device 6000 according to one embodiment. This schematic illustration depicts the main components of a testing device 6000 as shown in fig. 20-52. The testing device 6000 is an integrated device (i.e., the modules are housed within a single housing) that is suitable for use within a bedside facility (e.g., a doctor's office, pharmacy, etc.), a distributed testing facility, or in the home of the user. In some embodiments, the device 6000 can have a size, shape, and/or weight such that the device 6000 can be carried, held, used, and/or operated in a user's hand (i.e., it can be a "handheld" device). The handheld device may have a size of less than 15cmx15cmx15cm, or less than 15cmx15cmx10cm, or less than 12cmx12cmx6 cm. In other embodiments, the test device 6000 may be a self-contained single-use device. Similarly, the test device 6000 is a stand-alone device that includes all the necessary substances, mechanisms, and components to perform any of the molecular diagnostic tests described herein. Thus, the device 6000 does not require any external instruments to manipulate the biological sample, and only needs to be connected to a power source (e.g., to an a/C power source, coupled to a battery, etc.) to complete the methods described herein. In some embodiments, the testing device 6000 may be configured with a lock or other mechanism to prevent or attempt to reuse the device.

Furthermore, in some embodiments, the device 6000 may be a CLIA-exempt device and/or may operate according to a CLIA-exempt method. Similarly, in some embodiments, the device 6000 (as well as any of the other devices shown and described herein) is configured to operate in a sufficiently simple manner and can produce sufficiently accurate results to create a limited likelihood of misuse and/or a limited risk of harm when improperly used. In some embodiments, the device 6000 (as well as any of the other devices shown and described herein) can be operated by a user with little (or no) scientific training, in accordance with methods that require little judgment by the user and/or in which certain operating steps are easily controlled and/or automatically controlled. In some embodiments, the molecular diagnostic test device 6000 may be configured for long-term storage in a manner that creates a limited likelihood of improper use (reagent damage, reagent expiration, reagent leakage, etc.). In some embodiments, the molecular diagnostic test device 6000 is configured to be stored for up to about 36 months, up to about 32 months, up to about 26 months, up to about 24 months, up to about 18 months, up to about 6 months, or up to any value therebetween.

The testing device 6000 is configured to operate the biological sample S1 to generate one or more output signals associated with the target cells. Specifically, the apparatus 6000 includes a sample preparation module 6200, a fluid drive (or fluid transfer) module 6400, an amplification module 6600, a detection module 6800, a reagent module 6700, a valve 6300, and a control module (not shown). The test device and certain components thereof may be similar to any of the molecular test devices shown and described in international patent publication No. WO2016/109691 (which is incorporated herein by reference in its entirety) herein or entitled "device and method for molecular diagnostic testing". Accordingly, a detailed description of certain modules (e.g., the fluid drive module 6400) is not provided herein. A description of each module is provided below.

FIGS. 20-53C show various views of a molecular diagnostic test device 6000. The testing device 6000 is configured to manipulate the input sample to produce one or more output signals associated with the target cells according to any of the methods described herein. The diagnostic test device 6000 includes a housing 6001 (including an upper portion 6010 and a bottom portion 6030) within which housing 6001 the modules described herein are housed in whole or in part. In other words, the enclosure 6001 (including the upper portion 6010 and/or the bottom portion 6030) at least partially surrounds and/or encloses the module. Fig. 22-25 are various views showing a sample preparation module 6200, a fluid drive (or fluid transfer) module 6400, an amplification module 6600, a detection module 6800, a reagent module 6700, a fluid transfer valve 6300, and an electronic control module 6900 located within a housing 6001. A description of each module and/or subsystem follows the description of the housing assembly 6001.

Housing assembly 6001 includes upper housing 6010, bottom housing 6030, and lid 6050 (which functions as a cover and an actuator). As shown, the upper housing 6010 defines a detection opening (or window) 6011 and a series of status indicator light openings 6012. Upper housing 6010 also includes a sample input portion 6020 and a label 6013. The status indicator light opening 6012 is aligned with one or more light output devices (e.g., LEDs) of the electronic control module 6950. In this manner, the light output produced by the status indicator light is visible through the status indicator light opening 6012. Such light output may indicate, for example, whether the device 6000 has received power from a power source, whether an error has occurred (e.g., an error associated with an insufficient sample volume, etc.), and whether the test has been successfully completed.

The detection opening (or window) is aligned with the detection module 6800. In this way, the signal generated by and/or on each detection surface of the detection module 6800 is visible through the detection opening 6011. In some embodiments, upper housing 6010 and/or label 6013 are opaque (or translucent), thereby "framing" or protruding the detection opening. In some embodiments, for example, upper housing 6010 may include markings (e.g., bold lines, color, etc.) to highlight detection opening 6011. For example, in some embodiments, the upper shell 6010 may include a marker 6014 that identifies the detection opening as a specific disease (e.g., Chlamydia Trachomatis (CT), Neisseria Gonorrhoeae (NG), and Trichomonas Vaginalis (TV)) control. In other embodiments, upper housing 6010 need not include detection opening 6011. For example, in such embodiments, the signal generated by detection module 6800 is not visible to the naked eye, but is read out using another method. For example, in some embodiments, the readout may include scanning or otherwise receiving the signal OP1 using an auxiliary device, such as a mobile computing device. In still other embodiments, the reading the result can include indirectly reading a secondary signal that conveys a result related to (or descriptive of) the primary output from the detection module 6800.

Referring to fig. 26 and 27, the sample input portion 6020 includes a set of guide rails 6023 and lock grooves 6024, both of which are disposed on a bottom (or interior) surface of the upper housing 6010. The sample input portion 6020 also defines a sample input opening 6021 and an actuator opening 6022. The sample input opening 6021 aligns with the input opening 6212 (of the sample preparation module 6200) and provides an opening through which the biological sample S1 may be transferred into the device 6000. In addition, the sample input portion also allows a cover (or actuator) 6050 to be movably connected with the upper housing 6010. Specifically, as shown in fig. 20, 33, and 34, the lid 6050 is connected to the upper housing 6010 such that the handle 6070 of the actuator extends through the actuator opening 6022. The actuator opening 6022 is elongated to allow sliding movement of the lid 6050 relative to the upper housing 6010, as described herein. In addition, the guide rails 6023 are coupled to corresponding guide slots 6055 (see fig. 28 and 29) of the cover 6050 to facilitate sliding movement of the cover 6050. As shown in fig. 33 and 34, the locking groove 6024 of the upper housing 6010 is configured to receive the locking projection 6072 (see fig. 28 and 29) of the lid 6050 when the lid 6050 is in the second (or closed) position to prevent movement of the lid. In this manner, the upper housing 6010 includes a locking mechanism that retains the lid 6050 in its second (or closed) position to prevent reuse of the diagnostic device 6000, transfer of additional sample into the device 6000, or attempt to actuate the lid 6050 multiple times.

Lower housing 6030 includes a floor 6031 and defines a volume within which modules and/or components of apparatus 6000 are disposed. As shown in fig. 26, the bottom plate 6031 defines a series of flow channels 6035 that align with the flow channels of other components within the apparatus 6035 to allow fluid transfer between individual modules and components without the need for tubing, clamps, or the like. Specifically, as shown in fig. 45, the bottom of the reagent module 6700 defines a series of flow channels 6735, the flow channels 6735 corresponding to the flow channels 6035 in the bottom plate 6031, thereby facilitating the transport of fluid within the device. As shown in fig. 26, the lower housing 6030 defines an opening 6038, the opening 6038 being aligned with the power input of the electronic control module 6950. In use, one end of the power cord can be connected to the electronic control module 6950 through the opening 6038 (see, e.g., connection of power cord 6905 in fig. 53C).

As shown in fig. 28-30, the lid 6050 includes a first (or outer) surface 6051 and a second (or inner) surface 6052. Referring to fig. 33 and 34, a cover 6050 is attached to the housing 6001 and is positioned between the upper housing 6010 and the flexible panel 6080. As described below, the lid 6050 and flexible plate 6080 collectively actuate the reagent module 6700 as the lid 6050 moves relative to the housing 6001. As shown in fig. 30, interior surface 6052 defines a pair of channels 6055 and includes a pair of rails 6056. As described above, channels 6055 connect to corresponding rails 6023 of housing 6001 to facilitate sliding movement of lid 6050. The rails 6056 of the lid 6050 are configured to engage the flexible plate 6080, thereby facilitating sliding movement of the lid 6050 (relative to the flexible plate 6080). As shown by arrow GG in fig. 34, lid 6050 is configured to move relative to enclosure 6001 from a first (or open) position (fig. 33) to a second (or closed) position (fig. 34).

Similar to the lid 2050 described above, the lid 6050 is configured to perform multiple functions when moved relative to the housing 6001, thereby facilitating actuation of the device 6000 by a single motion. Specifically, the lid 6050 includes a sealing portion 6053, a switching portion 6060, and three reagent actuators 6064. The seal 6053 (also referred to as a cover) includes a cover surface 6057 and defines an input opening 6054. When the lid 6050 is in the open position (see, e.g., fig. 20,21 and 53A), the input opening 6054 is aligned with each of the sample input opening 6021 of the upper housing 6010 and the input opening 6212 of the sample preparation module 6200, and thereby provides an opening through which the biological sample S1 may be transferred into the apparatus 6000. Cover surface 6057 is a flat surface that covers (or blocks) each of sample input opening 6021 and input opening 6212 of upper housing 6010 when the lid is in the closed position (see fig. 53B and 53C). Specifically, when the lid 6050 is in the open position, the cover surface 6057 is spaced from the input opening 6212 and/or the sample input opening 6021, but when the lid 6050 is in the closed position, it covers the input opening 6212 and/or the sample input opening 6021. In some embodiments, the seal 6053 and/or the cover surface 6057 comprise a seal, gasket, or other material to fluidly isolate the sample input volume 6211 (of the sample preparation module 6200) when the lid 6050 is in the closed position.

In addition to covering the input opening 6212, closing the lid 6050 actuates other mechanisms within the device 6000. Specifically, as shown in fig. 29 and 30, the switch portion 6060 includes a protrusion that actuates the switch 6906 when the lid 6050 is moved from the open position to the closed position. When the switch is actuated (i.e., moved from the first state to the second state), power from a power source (e.g., power source 6905) can be provided to the electronic control module 6950 and any other components within the device 6000 that require power for operation. For example, in some embodiments, power is provided to either heater (e.g., heater 6230 of sample preparation module 6200, heater 6630 of amplification module 6600, and heater 6840 of detection module 6800) directly or through electronic control module 6950. This allows the heater 6230 to begin preheating for the cracking operation, for example, after the lid 6050 is closed, and the device 6050 is coupled to the power supply 6905 without further user action. Although the switch 6906 is shown as a rocker switch actuated directly by a protrusion of the switch portion 6060, in other embodiments, the switch 6906 (and corresponding switch portion 6060) can be any suitable switch that performs the functions described herein. For example, in some embodiments, the switch may be an insulating element that electrically insulates the power supply 6905 from the remaining components of the electronic control module 6950. In such embodiments, the switch portion 6060 can be connected to the insulating member and the insulating member can be removed (thereby electrically connecting the power supply 6905 to the electronic control module 6950). In other embodiments, the switch portion 6060 is an insulating element and no separate switch is included in the electronic control module 6950.

Referring to fig. 30 and 31, the reagent actuator 6064 includes a series of ramps that apply an actuation force to a corresponding set of deformable actuators 6083 of the flexible plates 6080 as the lid 6050 is moved from the open position (fig. 33) to the closed position (fig. 34). In this manner, the reagent actuator 6064 (and the deformable actuator 6083 of the flexible plate 6080) releases reagent from the sealed reagent container within the reagent module 6700, as described in more detail below.

An outer surface 6051 of the lid 6050 includes a handle 6070 and a locking protrusion 6072. The handle 6070 extends through the actuator opening 6022 of the upper housing 6010 and provides a structure that can be manipulated by a user to move the lid 6050 from an open position to a closed position. The locking protrusion 6072 has an inclined (or angled) protrusion that is held in sliding contact with the inner surface of the upper casing 6010 (see the inner surface shown in fig. 27). Because the slope of the locking projection 6072 is sharply angled, the locking projection can be moved in the direction indicated by arrow GG in fig. 34 to close the lid 6050. In addition, the continued contact between the locking protrusion 6072 and the upper housing 6010 prevents unintended closing of the lid 6050 by providing some resistance (i.e., friction) to the lid closing. As shown in fig. 34, when the lid 6050 is in the closed position, the locking projections 6072 are received within the locking slots 6024 of the upper housing 6010. The surface of the locking protrusion 6072 opposite the ramp forms a substantially 90 degree angle, thereby preventing movement of the lid 6050 in the opposite direction when the locking protrusion 6072 is within the groove 6024. In this manner, the lid 6050 is irreversibly locked after being closed, thereby preventing reuse of the apparatus 6000 and/or addition of supplemental sample fluid.

Flexplate 6080 (shown in fig. 31 and 32) includes an outer surface 6081 and an inner surface 6082. As described above, the cover 6050 is movably disposed between the upper housing 6010 and the flexible plate 6080. In other words, the outer surface 6051 of the cover 6050 faces the inner surface of the upper housing 6010, and the inner surface 6052 of the cover 6050 faces the outer surface 6081 of the flexible board 6080. The flexible plate includes three deformable actuators 6083, each aligned with a corresponding reagent actuator 6064 of the lid 6050 and one of the reagent containers 6701,6702, 6703. Thus, as the lid 6050 moves relative to the housing 6001, the reagent actuator 6064 and deformable actuator 6083 actuate the reagent module 6700. In particular, as described in detail below, the reagent actuator 6064 and the deformable actuator 6083 move the reagent container 6701,6702,6703 within the reagent shunt 6730 to release the reagent sealed within the container.

The flex plate 6080 defines a channel 6084 for surrounding at least three sides of each deformable actuator 6083. Thus, each deformable actuator 6083 remains connected to the flexible plate 6080 through a small band of material (or living hinge) 6085. Thus, when the reagent actuator 6064 applies an inward force to the outer surface 6086 of the deformable actuator 6083, the deformable actuator flexes or deforms inward toward the reagent module 6700, as indicated by arrow HH in fig. 34. This action causes the inner surface 6087 of each deformable actuator 6083 to apply an inward force to the reagent container (and deformable support member 6770), thereby causing the reagent container to move downward within the reagent shunt 6730, as indicated by arrow HH in fig. 34.

Referring to fig. 33, 34, 44 and 45, the reagent module 6700 includes a reagent shunt tube (or housing) 6730, three reagent containers 6701,6702,6703 and a deformable support member 6770 (see fig. 35 and 36). The reagent module 6700 provides a mechanism for long-term preservation of reagents within sealed reagent containers, actuation of the reagent containers to release the reagents from the reagent containers for use during the methods described herein. In addition to providing preservation and actuation functions, the reagent module 6700 also provides fluidic interconnections to allow reagents and/or other fluids to be transferred within the device 6000. In particular, as described herein, the reagent module 6700 is fluidly connected to the fluid transfer valve 6300 in a manner that allows for selective venting, fluid connection, and/or delivery of reagents and substances within the device 6000.

The reagent module 6700 holds packaged reagents, identified herein as reagent R4 (dual purpose blocking and washing solution), reagent R5 (enzymatic reagent), and reagent R6 (substrate), and allows for easy opening of the package and use of these reagents in the detection module 6800. As schematically shown in fig. 19, the reagent module 6700 comprises a first reagent container 6701 (containing reagent R4), a second reagent container 6702 (containing reagent R5) and a third reagent container 6703 (containing reagent R6). Each reagent container includes a connector at a first end and a frangible seal at an opposite second end. Specifically, as shown in fig. 33 and 34, the first reagent container 6701 includes a connector 6712 and a frangible seal 6713. The connector 6712 connects the first reagent container 6701 to the mating connection portion 6775 of the deformable support member 6770. The frangible seal 6713 is any suitable seal such as, for example, a heat-sealed BOPP film (or any other suitable thermoplastic film). Such films have excellent barrier properties, preventing interaction between the fluid inside the reagent container and external moisture, and have fragile structural properties that allow the film to be easily broken when needed. When the reagent container is pushed into the piercer, as described below, the frangible seal breaks, allowing liquid reagent to flow into the appropriate reagent reservoir when vented by the fluid transfer valve 6300. Although only the details of the first reagent container 6701 are shown and described herein, the second reagent container 6702 and the third reagent container 6703 have similar structures and functions.

Referring to fig. 44 and 45, the reagent shunt 6730 includes an upper (or outer) surface 6731 and a bottom (or inner) surface 6732. The reagent shunt 6730 includes three reagent canisters extending from the upper surface 6731 and having reagent containers disposed therein. Specifically, the reagent bypass tube includes a first reagent tank 6741 in which a first reagent container 6701 is disposed, a second reagent tank 6742 in which a second reagent container 6702 is disposed, and a third reagent tank 6743 in which a third reagent container 6703 is disposed. The reagent housing 6730 includes a pair of puncturers at a bottom portion of each reagent tank. The puncturers are configured to puncture the frangible seal of each reagent container as the reagent container is moved downwardly within the reagent housing 6730. In other words, the reagent housing 6730 includes a set of puncturers that puncture the respective frangible seal to open the respective reagent container when the reagent module 6700 is actuated. Referring to fig. 33 and 34, by way of example, the reagent housing 6730 includes a set of puncturers 6754 within a first reagent tank 6741. The reagent housing 6730 includes similar puncturers in the second reagent tank 6742 and the third reagent tank 6743. Furthermore, the piercer defines a flow path that places the internal volume of the reagent container and/or reagent canister in fluid communication with the outlet of the reagent module 6700 after the frangible seal is pierced.

The deformable support member 6770 includes an outer surface 6771 and an inner surface 6772. As described above, the outer surface 6771 includes an actuation zone that is aligned with one of the deformable actuators 6083 of the flexplate 6080. The inner surface 6772 includes three sealing portions 6773 and three connecting portions 6775. As shown in fig. 33 and 34, each seal portion 6773 is connected to the reagent housing 6730 to fluidly isolate the interior volume (i.e., reagent reservoir) of the respective reagent tank. The connection portions 6775 are each connected to one connector of the corresponding reagent vessel. As an example, one of the sealing portions 6773 is connected to the top of the first reagent tank 6741 to fluidly isolate (or seal) the interior volume of the first reagent tank 6741. In addition, one of the connection portions 6775 is connected to the connector 6712 of the first reagent container 6701.

The deformable support member 6770 is configured to deform from a first configuration (fig. 33) to a second configuration (fig. 34) in response to an actuation force applied thereto (e.g., by deformable actuator 6083). In addition, the deformable support member 6770 is biased in the first (or undeformed) configuration. In this manner, the deformable support member 6770 supports each reagent container in a "stored state" when the deformable support member 6770 is in the first configuration. In other words, when the deformable support member is in the first configuration, the deformable support member 6770 maintains the piercer 6754 spaced from the frangible seal 6713 of the reagent container 6701.

As the cover 6050 is moved, the downward force applied by the deformable actuator 6083 causes the deformable support member 6770 to transition to the second (or deformed) configuration (fig. 34). In other words, when the downward force is sufficient to overcome the opposing biasing force of the deformable support member 6770, the deformable support member 6770 transitions to the second configuration, as shown by arrow HH in fig. 34. This causes each reagent container to move downwardly within the corresponding reagent tank, bringing the piercer into contact with the frangible seal of each reagent container. In other words, when the deformable support member 6770 is in the second configuration, the puncturer 6754 punctures the frangible seal 6713 of the reagent container 6701, thereby releasing the reagent R4 from within the reagent container 6701. Although fig. 34 shows that only the first reagent container 6701 is actuated when the reagent module 6700 is actuated, each of the first, second, and third reagent containers 6701,6702,6703 are actuated in this manner. Thus, closing the lid 6050 actuates all reagent containers in addition to covering the sample input opening and providing power to the electronic control module 6950.

Although shown as including three reagent containers, in other embodiments, the reagent module 6700 (or any of the reagent modules described herein) can have any suitable number of reagent containers. For example, in some embodiments, a reagent module may include only one reagent container, like reagent module 2700 described herein.

Referring to fig. 44, the outer surface 6731 of the reagent shunt 6730 includes a set of valve fluid interconnects 6736, a set of mixing chamber fluid interconnects 6737, and a set of detection module fluid interconnects 6738. Each of these fluid interconnects is connected to one of the reagent tanks and/or other components within the device 6000 through a flow channel 6735 defined in the inner surface 6732. In addition, the outer surface 6731 includes a plurality of mounting clips 6790. Thus, the valve fluid interconnect 6736 (and appropriate passages 6735) provide a fluid connection with the fluid transfer valve 6300, and the fluid transfer valve 6300 is connected with the upper surface 6731 by one of the clips 6790. The mixing chamber fluid interconnect 6737 (and appropriate channels 6735) provide a fluid connection with a mixing assembly 6250, the mixing assembly 6250 being connected with the upper surface 6731. The detection module fluid interconnect 6738 (and appropriate channels 6735) provide a fluid connection with the detection module 6800.

FIGS. 37-41 show various views of a sample preparation module 6200. As described herein, the sample preparation (or fractionation) module 6200 may perform any or all of the following: A) receiving the biological sample S1, B) mixing the biological sample with desired reagents (e.g., a positive control reagent R1 and a reverse transcriptase R2), C) performing a lysis operation to release target RNA from the biological sample S1, D) performing a reverse transcription reaction to generate cDNA, and E) heating the resulting solution to inactivate the reverse transcriptase. Thus, in some embodiments, the sample preparation module is capable of performing efficient rapid RT-PCR within a single environment or module. By eliminating the need for external sample preparation and cumbersome instrumentation, the device 6000 is suitable for use in a bedside facility (e.g., a doctor' S office, pharmacy, etc.) or in the home of the user and can receive any suitable biological sample S1. Biological sample S1 (and any of the input samples described herein) can be, for example, blood, urine, a male urethral specimen, a vaginal specimen, a cervical swab specimen, and/or a nasal swab specimen collected using a commercially available sample collection kit.

The sample preparation module 6200 includes an upper body portion 6201, a bottom body portion 6202, a heater 6230, and a mixing assembly 6250. The upper body 6201 and the bottom body 6202 may be collectively referred to as a sample preparation housing, a flow member, or a reverse transcription chamber. While the flow member is shown as being constructed of two pieces (the upper body portion 6201 and the bottom body portion 6202) connected together, in other embodiments, the flow member may be of unitary construction. The sample preparation housing (i.e., the upper body portion 6201 and the bottom body portion 6202) defines a sample input opening 6212, a first (or retention) volume 6211, and a serpentine flow channel 6214. In some embodiments, the upper body portion 6201 and/or the bottom body portion 6202 may define one or more vents. Such vents can allow air to flow into or out of the sample preparation module 6200 (including the first volume 6211 and the serpentine flow channel 6214) as sample is transferred into and/or out of the sample preparation module 6200. Additionally, the upper body 6201 includes a set of fluid interconnects 6215, said fluid interconnects 6215 allowing the sample preparation module 6200 to be fluidically connected to the fluid transfer valve 6300 and other components within the device 6000.

The sample input opening 6212 is an opening into which the first (or retention) volume 6211 can enter. As described above, the biological sample S1 can be transferred into the retention volume 6211 through the sample input opening 6212 when the lid 6050 is in the open position. The first (or retention) volume 6211 is a volume within which the biological sample S1 can be mixed with reagents and also heated. For example, in some embodiments, the biological sample S1 can be collected in the holding volume 6211 and mixed with either or both of a control organism (identified as reagent R1) and a reverse transcriptase (identified as reagent R2). The control organism and the reverse transcriptase can each be lyophilized or otherwise in solid form. Furthermore, the reagents R1 and R2 may be secured within the retention volume 6211 to prevent the reagents R1 and R2 from inadvertently falling out of the device 6000, for example during storage, transportation or use. For example, in some embodiments, the reagents can be immobilized within the retention volume 6211 by a cover, basket, or other structure within the retention volume 6211.

In some embodiments, the agent R1 is a positive control organism, such as vibrio fischeri (alivibrio fischeri), luteinil netherlands (n.subflava), or any other suitable organism. In particular, Vibrio fischeri is suitable because it is gram-negative, nonpathogenic, bio-safe to grade 1, environmentally friendly, and highly unlikely to be found on humans. The positive control surface within the detection module contains capture probes for both the control organism (e.g., vibrio fischeri) as well as each target organism. This configuration ensures that the positive control surface always produces color if the device is working properly. If only the control organisms are present, a very strong positive for one of the target organisms during PCR may "wash out" or "outweigh" the amplification for the control organism. In this case, the positive control sample will not produce a color change, which can be confusing to the user. This configuration facilitates a user with little (or no) scientific training to operate the detection method and apparatus 6000 in a manner that requires little judgment.

In some embodiments, reagent R2 comprises a reverse transcriptase and other components that facilitate the RT-PCR methods described herein. For example, in some embodiments, reagent R2 includes salts that are required to establish the correct buffer environment for RT-PCR. The reagent R2 is formulated to dissolve in the biological sample within the retention volume 6211.

The biological sample may be heated within the retention volume 6311 to lyse cells within the biological sample S1 and further lyse (or release) target RNA from any viruses contained within the biological sample S1. In other words, the biological sample S1 may be heated to destroy the cells and also to destroy viruses in the cells to release the target RNA for detection. In particular, the heater 6230 is coupled to the sample preparation housing and/or the bottom body portion 6202 such that a first portion of the heater 6230 can transfer thermal energy into the retention volume 6211. The first portion of the heater 6230 may maintain the biological sample S1 at any suitable temperature for any of the time periods described herein. For example, in some embodiments, the biological solution can be maintained at a temperature within the lysis temperature range to release ribonucleic acid (RNA) molecules. The cracking temperature may range, for example, from about 25C to about 70C. In other embodiments, the cracking temperature may range from about 25C to about 50C.

Referring to fig. 39, which illustrates a top cross-sectional view of the sample preparation housing, the first volume 6211 is in fluid communication with the serpentine flow channel 6214 through the inlet opening 6213. In this way, the lysed biological sample (also referred to as a reverse transcription solution) mixed with the RT enzyme can flow from the first (or retention) volume 6211 through the serpentine flow channel 6214. More specifically, when a pressure gradient is applied across the inlet and outlet ports 6213, 6215 (e.g., by the fluid drive module 6400), reverse transcription solution can flow from the retention volume 6211 (the first volume) through the serpentine flow channel 6214. The serpentine channel provides a high surface area to volume ratio, thus allowing rapid RT-PCR and inactivation of the cleavage and/or RT enzymes in solution.

In use, the reverse transcription solution can be heated while flowing through the serpentine flow channel 6214 to perform RT-PCR and further inactivate the enzyme. In particular, the heater 6230 is attached to the sample preparation housing and/or the bottom body portion 6202 such that a second portion of the heater 6230 can transfer thermal energy into the serpentine flow channel 6214. The second section of the heater 6230 may maintain the reverse transcription solution at any suitable temperature and for any period of time described herein. For example, in some embodiments, the reverse transcription solution can be maintained at a temperature within the reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules. By rapidly progressing to reverse transcription, the residence time of released RNA in the reverse transcription solution can be minimized. Reducing the residence time may reduce the likelihood of degradation of the released RNA by ribonucleases (rnases). Limiting this potential degradation by performing lysis and RT-PCR in a single environment can reduce inconsistencies due to variations in RNA degradation. In addition, the rapid and single-context approach enabled by the sample preparation module 6200 may allow the RT-PCR methods described herein to be performed without the use of ribonuclease inhibitors and/or on unfiltered samples. The reverse transcription temperature may range, for example, from about 30C to about 80C. In other embodiments, the reverse transcription temperature may range from about 50C to about 60C.

In addition to enabling rapid RT-PCR, the sample preparation module 6200 may also heat the reverse transcription solution to a temperature sufficient to inactivate one or more cleavage or RT enzymes contained therein. For example, the heating element can heat the reverse transcription solution within the channel 6214 to about 57 ℃, about 58 ℃, about 59 ℃, about 60 ℃, about 61 ℃, about 62 ℃, about 63 ℃, about 64 ℃, about 65 ℃, about 66 ℃, about 67 ℃, about 68 ℃, about 69 ℃, about 70 ℃, about 71 ℃, about 72 ℃, about 73 ℃, about 74 ℃, about 75 ℃, about 76 ℃, about 77 ℃, about 78 ℃, about 79 ℃, about 80 ℃, about 81 ℃, about 82 ℃, about 83 ℃, about 84 ℃, about 85 ℃, about 86 ℃, about 87 ℃, about 88 ℃, about 89 ℃, about 90 ℃, about 91 ℃, about 92 ℃, about 93 ℃, about 94 ℃, about 95 ℃, about 96 ℃, about 97 ℃, about 98 ℃, about 99 ℃, about 100 ℃ or greater than 100 ℃. The enzyme may be inactivated by heating the reverse transcription solution to an elevated temperature. In some embodiments, the sample may be heated to about 95C for about 4 minutes.

As described above, the flow member is in contact with the heating element 6230, which may be, for example, a Printed Circuit Board (PCB) heater. The heating element 6230 includes a connector 6231 and a plurality of segmented portions, and thus can independently generate thermal energy into the retention volume 6211 and the serpentine flow channel 6214. In some embodiments, the heating element 6230 is designed to heat the serpentine portion 6214 of the sample preparation module 6200 without heating the retention volume 6211, and vice versa.

To minimize thermal energy that may be unintentionally transferred between the retention volume 6211 and various portions of the serpentine channel 6214, or even between different portions of the serpentine channel 6214, one or more slots 6232 may be cut in the PCB6330 to isolate various portions of the heater 6230. For example, in some embodiments, the heater 6230 can comprise a series of slots and/or openings, as described in U.S. patent publication No. 2017/0304829 entitled "printed circuit board heater for amplification module," which is incorporated by reference herein in its entirety. Further, in some embodiments, the heating element of heater 6230 is located on the inner layer such that the top copper cladding (not shown) can act as a heat sink to minimize temperature variations along the serpentine path.

The reverse transcription solution, after flowing through the inactivation process, can flow through the flow control valve 6300 via outlet 6215 and into inlet 6217 of mixing assembly 6250. Mixing assembly 6250 mixes the output from serpentine flow channel 6214 with reagents (identified as R3) to perform a successful amplification reaction. In other words, the mixing module 6250 is configured to reconstitute the reagent R3 at a predetermined input volume while ensuring a uniform local concentration of the reagent R3 throughout the volume. In some embodiments, the mixing assembly 6250 is configured to generate and/or deliver a sufficient volume of liquid for the amplification module 6600 to provide a sufficient volume of output to the detection module 6800.

Referring to fig. 40 and 41, the mixing assembly 6250 is attached to the upper body 6201 and includes a bottom housing 6251, a head housing 6260, and a vibration motor 6265. The bottom housing 6251 defines a mixing reservoir 6255 and contains therein an amplification reagent R3. The bottom housing 6251 includes an inlet connector 6252 and an outlet connector 6253 and is attached to the upper body portion 6201 by support members 6254. An upper housing 6260 surrounds the mixing reservoir 6255 and provides a surface to mount the vibration motor 6265. The inlet connector 6252, the outlet connector 6253, and the support member 6254 may be constructed of any suitable materials and may have any suitable dimensions. For example, in some embodiments, the inlet connector 6252, the outlet connector 6253, and the support member 6254 are configured to limit the amount of vibrational energy from the motor 6265 that is transmitted into the remainder of the sample preparation module 6200. For example, in some embodiments, the inlet connector 6252, the outlet connector 6253, and/or the support member 6254 can be constructed of resilient or elastomeric materials to allow vibratory movement of the bottom and upper housings 6251, 6260 while transmitting this energy to the upper body 6201.

After mixing within the mixing assembly 6250, the prepared sample is then transferred to the amplification module 6600. The transfer of fluids (including reverse transcription solutions, reagents, etc.) is caused by the fluid drive (or transfer) module 6400. The fluid drive (or transmission) module 6400 may be a pump or series of pumps configured to generate a pressure differential and/or flow of the solution within the diagnostic test device 6000. In other words, the fluid transfer module 6400 is configured to produce fluid pressure, fluid flow, and/or otherwise transport biological samples and reagents through the various modules of the device 6000. The fluid transfer module 6400 is configured to contact and/or receive a sample stream therein. Thus, in some embodiments, the device 6000 is specifically configured for a single use to eliminate the possibility of contaminating the fluid transfer module 6400 and/or the possibility that the sample preparation module 6200 may be contaminated by a previous round of experimentation (thereby negatively affecting the accuracy of the results). As shown, the fluid transfer module 6400 may be a piston pump that is connected to the reagent module 6700 by a clip 6790. The fluid driver module 6400 may be driven and/or controlled by the electronic control module 6950. For example, in some embodiments, the fluid drive module 6400 may include a DC motor, the position of which may be controlled using a rotary encoder (not shown). In other embodiments, the processor 6951 of the electronic control module 6950 can include code for implementing and/or configured to implement a closed-loop method of tracking motor position by monitoring current consumption of the motor, as described in international patent publication No. WO2016/109691, entitled "apparatus and method for molecular diagnostic testing," the entire contents of which are incorporated herein by reference.

The amplification module 6600 includes a flow member 6610, a heater 6630, and a heat sink 6690. The flow member 6610 can be any suitable flow member that defines a volume or series of volumes within which the prepared solution S3 can flow and/or be maintained to amplify a target nucleic acid molecule within solution S3. Heater 6630 may be any suitable heater or set of heaters connected to flow member 6610 that may heat the prepared solution within flow member 6610 to perform any of the amplification operations as described herein. For example, in some embodiments, the amplification module 6600 (or any of the amplification modules described herein) can be similar to the amplification module shown and described in U.S. patent publication No. 2017/0304829 entitled "printed circuit board heater for amplification module" (which application is incorporated herein by reference in its entirety).

In some embodiments, the flow member 6610 defines a single volume within which the prepared solution is held and heated, thereby amplifying the nucleic acid molecules within the prepared solution. In other embodiments, the flow member 6610 may define a "zig-zag" or serpentine flow path through which the prepared solution flows. In other words, the flow member 6610 defines a curved flow path such that the flow path intersects the heater 6630 at multiple locations. In this way, the amplification module 6600 can perform a "flow-through" amplification reaction in which the prepared solution flows through a plurality of different temperature regions.

The flow member 6610 (and any flow member described herein) may be constructed of any suitable material and may have any suitable dimensions to facilitate the desired amplification performance for a desired volume of sample. For example, in some embodiments, amplification module 6600 (and any of the amplification modules described herein) can perform 6000 amplifications (6000X) or more in less than 15 minutes. For example, in some embodiments, the flow member 6610 (and any flow member described herein) is constructed from at least one of a cyclic olefin copolymer or a graphite-based material. Such materials contribute to the desired heat transfer characteristics into the flow path. Further, in some embodiments, the flow member 6610 (and any flow member described herein) may have a thickness of less than about 0.5 mm. In some embodiments, the flow member 6610 (and any flow member described herein) can have a volume of about 150 microliters or more, and the flow can be such that at least 10 microliters of sample is amplified. In other embodiments, at least 20 microliters of sample is amplified by the methods and devices described herein. In other embodiments, at least 30 microliters of sample is amplified by the methods and devices described herein. In still other embodiments, at least 50 microliters of sample is amplified by the methods and devices described herein.

Heater 6630 may be any suitable heater or collection of heaters that can perform the functions described herein to amplify the prepared solution. In some embodiments, heater 6630 may establish a plurality of temperature zones through which the prepared solution flows, and/or heater 6630 may define a desired number of amplification cycles to ensure a desired detection sensitivity (e.g., at least 30 cycles, at least 34 cycles, at least 36 cycles, at least 38 cycles, or at least 60 cycles). The heater 6630 (and any of the heaters described herein) may have any suitable design. For example, in some embodiments, the heater 6630 may be a resistive heater, a thermoelectric device (e.g., a Peltier device), or the like. In some embodiments, the heater 6630 may be one or more linear "sheet heaters" arranged to pass the flow path through the heater at a plurality of different points. In other embodiments, the heater 6630 may be one or more curved heaters having a geometry corresponding to the flow member 6610 to create a plurality of distinct temperature zones in the flow path.

Although amplification module 6600 is generally described as performing a thermal cycling operation on a prepared solution, in other embodiments, amplification module 6600 can perform any suitable thermal reaction to amplify nucleic acids within the solution. In some embodiments, amplification module 6600 (and any of the amplification modules described herein) can perform any suitable type of isothermal amplification process, including, for example, loop-mediated isothermal amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA) useful for detecting a target RNA molecule, Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), reticulo-branch amplification (RAM), or any other type of isothermal process.

Detection module 6800 is configured to receive the output from amplification module 6600 and reagents from reagent module 6700 to produce a colorimetric change indicative of the presence or absence of a target organism in the initial input sample. The detection module 6800 also generates a colorimetric signal to indicate the overall proper operation of the test (positive and negative controls). In some embodiments, the color change induced by the reaction is easily readable and is binary, without the need to interpret shading or hue. The detection module 6800 can be similar to the detection module shown and described in International patent publication No. WO2016/109691, entitled "apparatus and method for molecular diagnostic testing" (which application is incorporated herein by reference in its entirety).

Referring to fig. 64 and 65, the detection module includes a cover, a detection housing 6810, and a heater 6840. The heater 6840 may be similar to any of the circuit board heaters described herein and the circuit board heaters shown and described in international patent publication No. WO2016/109691 (which is hereby incorporated by reference in its entirety) entitled apparatus and method for molecular diagnostic testing. The lid and the detection housing 6810 form a flow cell for detection. The housing 6810 defines a detection chamber/channel 6812, the detection chamber/channel 6812 having a sample inlet port 6814, a first reagent inlet/outlet port 6815, and a second reagent inlet/outlet port 6816. The sample inlet port 6814 is in fluid connection with the outlet of the amplification module 6600 and receives the amplified sample. The first reagent port 6815 and the second reagent port are connected to the reagent module 6700 by a fluid interconnect 6738. Thus, in use, a wash/blocking reagent (e.g., previously identified as R4) can be delivered into the detection channel 6812 through the first reagent port 6815 or the second reagent port 6816. Similarly, a detection enzyme (e.g., previously identified as R5) and a detection substrate (e.g., previously identified as R6) can be delivered into the detection channel 6812 through the first reagent port 6815 or the second reagent port 6816. Additionally, the first reagent port 6815 or the second reagent port 6816 can also be used to receive or flow waste or excess reagents from the first reagent port 6815 or the second reagent port 6816.

The detection channel 6812 is surrounded or defined by a surface 6820, which surface 6820 includes one or more detection surfaces 6821, and a non-detection surface 6826. The detection surface 6821 includes a series of capture probes to which target amplicons can bind as the detection solution flows across the detection surface 6821. The capture probe may be any suitable probe formulated to capture or bind the target amplicon. Specifically, in some embodiments, the detecting portion 6821 includes 5 detecting surfaces. Each detection surface is chemically modified to contain the desired configuration of capture probes. In particular, in some embodiments, the first detection surface may comprise hybridization probes specific for gonococci (NG). The second detection surface may comprise hybridization probes specific for Chlamydia Trachomatis (CT). The third detection surface may comprise hybridization probes specific for Trichomonas Vaginalis (TV). The fourth detection surface can include non-target probes for negative controls. The fifth detection surface may comprise hybridization probes for positive controls (vibrio fischeri, vitessella microflavus, etc.).

The non-detection surfaces 6826 may be those surfaces surrounding the detection surface 6821. As described above with respect to detection module 3800, in some embodiments, the entire surface 6820 (including detection surface 6821 and non-detection surface 6826) can be coated with a blocking solution as part of the methods described herein.

Fluid transfer valve 6300 is shown in fig. 19 (schematic) and 46. Fig. 47-52 illustrate the fluid transfer valve 6300 in several different operating configurations, with the flow (or vent) housing 6310 shown in transparent line so that the position of the valve disc 6320 can be seen. The fluid delivery valve 6300 includes a flow housing 6310, a valve body (or disk) 6320, a main housing 6330, and a motor 6340. The flow housing 6310 defines a valve pocket within which the valve disc 6320 is rotationally disposed. The flow housing 6310 includes flow structures defining at least 6 transfer (or vent) flow paths, shown in fig. 47-52. Specifically, the flow paths include a sample inlet flow path 6312, a sample outlet flow path 6313, an amplification flow path 6314, a washing solution (reagent R4) aeration flow path 6315, a detection enzyme (reagent R5) aeration flow path 6316, and a detection substrate (reagent R6) aeration flow path 6317. The flow housing 6310 includes connections where each transfer or vent flow path may be connected to a respective module by interconnects as described herein. Each of the flow connection/vent ports described above is open to the valve housing. In this way, as the valve body 6320 rotates about the center of the valve sleeve (as shown by arrow JJ), the channel 6321 of the valve body 6320 can connect each central port to the other ports, depending on their radial and angular positions. The use of multiple radii allows not only a single port at a time, but multiple ports to be fluidly connected at a time, depending on the configuration.

The valve assembly 6300 can be moved between various configurations depending on the angular position of the valve body 6320 within the valve sleeve. Fig. 47-52 illustrate the assembly in various different configurations. Fig. 47 shows the valve assembly 6300 in a home (or initial position) with the sample inlet flow path 6312 and sample outlet flow path 6313, and other fluid connection/vent ports closed. Fig. 48 shows the valve assembly 6300 in a first rotational position, in which the sample inlet flow path 6312 and the sample outlet flow path 6313 are open. With the valve assembly 6300 in the first position, actuation of the fluid drive module 6400 can generate a flow of biological sample into and through the serpentine channel 6214 and then to the mixing assembly 6250. In this manner, device 6000 can perform an RT-PCR method (e.g., method 50, or any other RT-PCR method) as described herein. In addition, the timing of valve actuation and supply of power to the fluid drive module 6400 (e.g., a pump) can be controlled by the electronic control module 6950 to maintain the flow rate through the sample preparation module 6200 (including the serpentine channel 6214) within a range that can achieve the desired performance of RT-PCR.

Upon completion of the mixing process within the mixing assembly 6250, the valve assembly 6300 may be moved further to a second position (not shown). When the valve is in the second position, amplification flow path 6314 is open (i.e., aligned with flow channel 6321), thereby allowing for the transfer of the mixed solution (i.e., after RT-PCR) that is delivered into amplification module 6600. The timing of valve actuation and the supply of power to the fluid drive module 6400 (e.g., a pump) may be controlled by the electronic control module 6950 to maintain the flow rate through the sample amplification module 6600 within a range that can achieve the desired performance of amplification. Further, with the valve assembly 6300 in the second position, continued actuation of the fluid drive module 6400 will cause the amplified solution to be delivered to the detection module 6800 and pass through the detection module 6800.

As described herein, the detection operation is accomplished by delivering a series of reagents to the detection module at specific times. Although closing the lid 6050 actuates the reagent module 6700 to open (or release) the reagents from their respective sealed containers, the reagents remain in the reagent module 6700 until needed in the detection module 6800. When a specific reagent is required, the rotary valve 6300 opens an appropriate vent channel (i.e., the washing solution vent channel 6315, the detection enzyme vent channel 6316, and the detection substrate vent channel 6317) to the reagent module 6700. Actuation of the fluid drive module 6400 applies a vacuum to the outlet port of the reagent module 6700 (through the detection module 6800), thereby delivering the selected reagent from the reagent module 6700 into the detection module 6800. Fig. 49 shows the valve assembly 6300 in a third rotational position, in which the detection enzyme vent flow path 6316 is open. With the valve assembly 6300 in the third position, actuation of the fluid drive module 6400 may generate a flow of detection enzyme (reagent R5) into the detection module 6800. Fig. 50 shows the valve assembly 6300 in a fourth rotational position, in which the wash solution (reagent R4) vent flow path 6315 is open. With the valve assembly 6300 in the fourth position, actuation of the fluid drive module 6400 may generate a flow of wash (or multipurpose wash/seal) solution (reagent R4) into the detection module 6800. Fig. 51 shows the valve assembly 6300 in a fifth rotational position, in which the detection substrate (reagent R6) vent flow path 6317 is open. With the valve assembly 6300 in the fourth position, actuation of the fluid drive module 6400 may generate a flow of substrate (reagent R6) into the detection module 6800. Fig. 52 shows the valve assembly 6300 in a final position, in which the vent flow path is closed.

As described with reference to the apparatus 3000, methods 30, and methods 40 described above, in some embodiments, the device 6000 can include a multi-purpose wash/blocking reagent (e.g., reagent R4), and can deliver a portion of the multi-purpose wash/blocking reagent into the detection module 6800 at separate times. Specifically, in some embodiments, according to the methods 30 or 40 described herein, the valve assembly 6300 may be first placed in the fourth position (fig. 50) and a portion of the multi-purpose wash/seal reagent may be transferred into the detection module 6800. Additionally, after a predetermined dwell time (e.g., 30 seconds), and with the valve assembly 6300 still in the fourth position, the motion of the fluid drive module 6400 may be reversed to draw the multi-purpose wash/lock reagent back into the reagent module 6700. The valve assembly 6300 may then be moved to a first position to begin processing of the biological sample.

The apparatus 6000 may be used to perform any of the methods described herein. Referring to fig. 53A-53C, to use the device, a biological sample S1 is first placed in sample input opening 6021 (e.g., using sample transfer pipettor 6110), as described above. The cover 6050 is then moved to the closed position as indicated by arrow KK in fig. 53B. As described above, closing the lid 6050 encloses the sample input volume 6211, actuates the electronic control module 6950 (and/or the processor 6951 included therein), and also actuates the reagent module 6700, as described above. The device 6000 is then plugged into an outlet via the power cord 6905 to couple the device 6000 to a power source. In this way, the device 6000 can be actuated in a single action (i.e., closing the lid) in addition to placing the sample S1 therein and inserting the device into the receptacle.

Detection of HIV-1RNA in blood of pricked finger using RT-PCR deviceMethod and apparatus for bedside testing

In some embodiments, device 6000 or any of the devices described herein can be used to perform an HIV-1RNA detection assay. The HIV-1RNA detection assay enables unskilled persons to detect self-collection of blood samples from a pricked finger using inexpensive, disposable, instrument-free devices at home or in the facilities of less developed countries. The use of this device has the potential to shift the diagnosis of acute or early HIV infection and antiretroviral therapy monitoring. In some embodiments, the molecular diagnostic test device includes an amplification and detection platform to enable the production of cDNA from viral RNA on the device. In some embodiments, the cDNA is amplified by a serpentine PCR module.

In some embodiments, the molecular diagnostic test device comprises an HIV-1RNA detection platform (also known as the RT enhancement platform (RTEP)). Some versions of the diagnostic test device consist of an input port, an inactivation chamber, a mixing chamber, two check valves, a PCR module and a detection module with the necessary reagent containers, a piston pump and a rotary valve. Fig. 15 and 19 each show two examples of RTEP versions that include sample preparation modules that can perform RT-PCR as described herein. In addition, the sample preparation module incorporates a reverse transcription step to allow for the processing of viral RNA. The RT step is in series with the rest of the process and can therefore be bypassed by firmware control for test groups that do not require it. Heating is provided by a separate independent heating circuit on the pyrolysis heater plate.

In use, plasma (or blood) is dispensed into the lysis chamber, and the syringe pump is activated to create a vacuum that flows the sample through the heated channel where viral lysis occurs, releasing the genomic RNA. The temperature in the channel was controlled at 92C to ensure denaturation of viral RNA and the sample fluid was held at this temperature for approximately 30 seconds. The sample fluid then proceeds through the check valve and into the mixing chamber, which retains several lyophilized beads (PCR master mix reagent and RT enzyme) that are hydrated by the sample fluid. The chambers were mixed by a small vibrating motor and the sample was then incubated at 55C to allow reverse transcription of viral RNA into cDNA, at which time the syringe pump would reverse direction and pressurize the mixing chamber to move the chamber contents through an additional heater at 95C, thereby inactivating the RT enzyme and activating the thermostable hot start DNA polymerase. The process then proceeds to a PCR and detection module as described in any of the patent applications or publications herein or incorporated herein.

In some embodiments, the methods and devices may include multiple primer sets to address the problem of significant variability of the HIV-1 genome. For example, the target sequence may include highly conserved regions of both genes, and the primer sets may both be included as part of a multiplex assay. In addition, the methods and devices may include primers for the MS2 RNA phage, MS2 RNA phage serving as a lysis and amplification control. Thus, the resulting multiplex assay will contain three primer sets, two corresponding to independently conserved regions of the HIV-1 genome, and one corresponding to the MS2 phage genome. As detailed below, one primer in each set will be used to cause a reverse transcription step for the one-step RT-PCR assay used herein.

In some embodiments, the methods and devices may include forward and reverse primers and TaqMan probes for two HIV-1 genes and a MS2 phage positive control (table 1). The forward primer was 5' biotinylated. Reverse primers were also used to cause a one-step RT-PCR reverse transcription reaction. The TaqMan probe with the indicated sequence has a FAM fluorophore at the 5 'end and a BHQ2 quencher at the 3' end.

TABLE 1 initial forward and reverse primers and TaqMan probe sequences for one-step multiplex RT-PCR assay.

In some embodiments, the optimized multiplex PCR assay may comprise a one-step multiplex Reverse Transcription (RT) -PCR assay that uses HIV-1 and MS2 phage reverse transcription PCR primers to cause cDNA synthesis. The methods and devices may include validated primer sets and optimized master mixes (master mix) containing both reverse transcriptase and thermostable DNA polymerase. In this way, the device can perform an "ultrafast" one-step multiplex RT-PCR assay to amplify armored rna (armored rna) templates corresponding to two HIV-1 genes and the MS2 positive control gene. The time required to generate cDNA from an RNA template before PCR is initiated is critical because it must not extend the overall sample-feedback, turnaround time beyond the 20 minute specification of the assay. Each of the three viral armored RNA templates was serially diluted individually in TE buffer, and each dilution was then subjected to pure one-step RT-PCR using a laboratory instrument programmed to perform an ultra-fast RT step to generate cDNA, followed by "fast" cycle PCR amplification of the cDNA.

In some embodiments, the multiplex RT-PCR assay is characterized by the following aspects: 1) when the armored RNAs corresponding to the amplicon sequences of the two HIV-1 genes and the MS2 phage genes that were determined were diluted into pooled EDTA plasma samples, the assay detected and identified these armored RNAs; and 2) the assay detects and identifies low concentrations of each HIV-1 armored RNA corresponding to the desired LoD in pooled EDTA plasma samples. To ensure the desired result, in some embodiments, the assay (or device) may include a separate proprietary RT primer. In some embodiments, the method may comprise increasing the temperature of the RT step to reduce RNA secondary structure.

One potential problem to be solved by the current devices and methods relates to the presence of PCR inhibitors in the plasma, including EDTA, heme, and IgG. In part, the sample preparation module and method avoids this problem by using a nucleic acid-binding nylon filter; since once bound, the nucleic acids may be washed with a buffer substantially free of plasma components and then eluted in a buffer substantially free of plasma components. The use of the MS2 phage treatment and amplification controls provides a sensitive measure of the presence of the inhibitor. If PCR inhibition persists, the thermal cleavage step may be prolonged and/or the assay may employ variants of thermostable DNA polymerases that are resistant to stool inhibitors, such as Omni Klentaq. If EDTA chelates Mg, which reduces PCR efficiency, the concentration of Mg in the PCR mastermix can be increased.

In some embodiments, a method of detecting HIV may comprise separating plasma. In particular, plasma is a preferred sample matrix for monitoring virological control of humans receiving treatment with ARV and for the detection of HIV-1RNA in acute/early HIV infection. It is understood that other sample types (e.g., dried blood spots) are acceptable alternatives at remote locations, and that viruses may also be found in other bodily fluids, including vaginal secretions and semen. However, in some embodiments, the devices and methods may include any suitable plasma separation module that employs any desired separation method.

In some embodiments, the method includes a step-by-step user guidance process that may be performed at home or in a remote, developing home facility. The operations include: (1) obtaining a needle prick finger blood by a user using a commercially available lancet; (2) placing blood into a plasma separation module either directly or using commercially available capillaries included in a kit; (3) automatically separating plasma from blood by a plasma separation module; (4) the user transfers plasma from the plasma separation module to the HIV molecular diagnostic device (sample input port) using a pipette included in the kit; (5) the user activates the device by pressing any number of buttons; and (6) the user records the results. In some embodiments, the device comprises a physically integrated plasma separation module (i.e., within the molecular diagnostic device).

Plasma volume is a function of the lancing finger blood input volume and separation efficiency. It is understood that the range of finger stick blood volume estimates is large, but at least one commercially available lancet (BD blue) is reported to obtain an average of 400ul of blood (ref). The needled finger blood may be collected using commercially available EDTA-coated capillaries, the contents of which may be placed into the plasma separation module input ports. The separation efficiency averaged-30% of the volume of the needled finger blood. Thus, assuming the expected LoD of the Click HIV-1 device is ≦ 200 viral copies/ml plasma and a plasma separation efficiency of 30%, the minimum input volume required to meet this LoD is 150ul of blood, which will yield 45ul of plasma containing 8 copies of HIV-1 virus at a plasma HIV-1 concentration of 200 copies/ml.

In some embodimentsThe method includes separating plasma using a superhydrophobic plasma separator similar to the model licensed to Drummond Scientific developed by the group of professor Changchun Liu, university, pennsylvania. Show such a mechanism in<Within 10min, 65ul of hemoglobin free PCR-compatible plasma was extracted from 200ul of EDTA-anticoagulated blood. In some embodiments, the separator may comprise a 1.5X 1X 0.3 inch wide disposable device and an inverted asymmetric polysulfone membrane (Plasma Separation membrane, Pall), which uses a clamshell-type housing to accommodate a superhydrophobic sample well in which a pricked finger blood is placed. This combination allows Red Blood Cells (RBCs) in the sample to settle away from the membrane, rather than passing through it, thereby preventing membrane blockage and providing a more efficient means of separation. Plasma is then collected at the plasma discharge port where it can be removed using a simple low pressure vacuum created by withdrawing the plunger of a close fitting pipette.

In some embodiments, the methods include separating plasma using a spiral glass-fiber membrane enclosed within a protective cassette that allows the separation of cellular components of blood from a cell-free plasma sidestream with minimal hemolysis. Such separation devices may include a HemaSpot-SE device that receives a small amount of a needled finger blood sample. When 4-5 drops of pricked finger blood (150. mu.L) were applied in the center of the device, a yield of 50. mu.L of plasma was produced, thereby providing a plasma separation efficiency of 33%, similar to that obtained by the superhydrophobic membrane described above. As part of the synergy presented herein, the current device would be modified to accept a blood volume of 150-. Once the needle-prick finger sample is applied to the input port, the cassette is closed. Within 3 minutes, the plasma separation was completed, the cassette was opened, and the still wet lower half of the spin-on filter, containing no blood, containing plasma, was removed and transferred to a capped tube containing the universal transport medium. The tube was vortexed to elute the virus from the membrane, and then the liquid was pipetted into the sample processing reservoir of the HIV-1 molecular diagnostic test device.

In some embodiments, the method does not require plasma separation, but selectively amplifies only HIV-1RNA (but not pre-viral DNA) in an ETDA anticoagulated sample in a molecular diagnostic test device.

Method and apparatus for detecting upper respiratory tract infection using RT-PCR device

In some embodiments, any of the devices described herein can be used to perform a single use (disposable), on-demand field diagnostic test for detecting influenza a (Flu a), influenza B (Flu B), and Respiratory Syncytial Virus (RSV) from a nasal swab specimen. This will help clinicians identify patients with better antiviral drug action, thereby reducing the prescription of unnecessary ineffective antibiotics that lead to antimicrobial drug resistance

In some embodiments, the test devices (and methods) may comprise a nasal swab and may be performed on any of the devices described herein.

In some embodiments, the methods and devices may be optimized to ensure that cross-reactivity with the following pathogens (listed in table 2) is limited.

TABLE 2 list of pathogens

In addition, assay performance can be optimized to avoid performance degradation in the presence of inanimate substances that may be present in infected nasal secretions (see list below) and that may interfere with device performance, including common topical nasal decongestants (Afrin), topical steroid nasal sprays (Flonase), and human whole blood and mucin. In some embodiments, each assay includes MS2 phage as a positive control that monitors the performance of the assay from the sample processing step via RT-PCR amplification to amplicon detection on the detection platform. If these or other substances inhibit any aspect of the performance of the assay, the positive control will register as "not detected" and the assay result will be inconclusive.

TABLE 3 list of pathogens

In some embodiments, any of the systems described herein can be modified to perform an enteropathogen diagnostic assay that simultaneously detects DNA bacteria (i.e., campylobacter jejuni (c. jejuni), salmonella enterica (s. enterica), Shigella (Shigella) species) and RNA viral targets (norovirus).

Although the amplification module is generally described herein as performing a thermal cycling operation on a prepared solution, in other embodiments, the amplification module may perform any suitable thermal reaction to amplify nucleic acids within the solution. In some embodiments, any of the amplification modules described herein can perform any suitable type of isothermal amplification process, including, for example, loop-mediated isothermal amplification (LAMP), Nucleic Acid Sequence Based Amplification (NASBA) useful for detecting a target RNA molecule, Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), reticulo-branch amplification (RAM), or any other type of isothermal process.

While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Where the above-described methods and/or schematics indicate particular events and/or flow patterns occurring in a particular order, the order of the particular events and/or flow patterns may be modified. While embodiments have been particularly shown and described, it will be understood that various changes in form and detail may be made.

For example, any of the sample input modules, sample preparation modules, amplification modules, heater assemblies, and detection modules shown and described herein can be used in any suitable diagnostic device. Such devices may include, for example, single-use devices that may be used at a bedside facility and/or in a user's home. In other words, in some embodiments, the apparatus (and any other apparatus shown and described herein) may be configured for use in a decentralized testing facility. Further, in some embodiments, any of the sample input modules, sample preparation modules, amplification modules, heater assemblies, and detection modules shown and described herein can be included within a CLIA-exempt device and/or can facilitate operation of the device according to the CLIA-exempt method. In other words, in some embodiments, the sample input module, sample preparation module, amplification module, and detection module shown and described herein can facilitate operation of the device in a manner that is simple enough to produce results that are accurate enough to create a limited likelihood of improper use and/or a limited risk of harm when improperly used. In some embodiments, the sample input module, sample preparation module, amplification module, and detection module shown and described herein can be used in any of the diagnostic devices shown and described in international patent publication No. WO2016/109691 (which is hereby incorporated by reference in its entirety) entitled "devices and methods for molecular diagnostic testing.

In some embodiments, any of the methods described herein, such as method 50 and the methods described with respect to fig. 17A-17C, can include the following time, temperature, and volume ranges provided in table 4.

TABLE 4 sample Range

The devices and methods described herein can be used to analyze any suitable type of biological sample, such as a tissue sample (e.g., a blood sample). In some cases, the biological sample comprises a bodily fluid taken from the subject. In some cases, the bodily fluid includes one or more cells comprising a nucleic acid. In some cases, the one or more cells include one or more microbial cells, including, but not limited to, bacteria, archaea, protists, and fungi. In some cases, the biological sample includes one or more viral particles. In some cases, the biological sample includes one or more microorganisms that cause sexually transmitted diseases. The sample may include a sample from a subject, such as whole blood; a blood product; red blood cells; (ii) a leukocyte; a white film layer; a swab; (ii) urine; sputum; saliva; semen; lymph fluid; endolymph fluid; perilymph fluid; gastric juice; bile; mucus; sebum; sweat; tear fluid; vaginal secretions; vomit; feces; breast milk; earwax; amniotic fluid; cerebrospinal fluid; effusion of the abdominal cavity; pleural effusion; a biopsy specimen; cyst fluid; synovial fluid; a vitreous humor; aqueous humor; a cyst fluid; eye wash; ocular aspirates; plasma; serum; lung lavage fluid; lung aspirates; animal tissue, including human tissue, including but not limited to liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell cultures and lysates, extracts or materials and fractions obtained from the above samples or any cells and microorganisms and viruses that may be present on or in the sample. The sample may comprise cells of a primary culture or cell line. Examples of cell lines include, but are not limited to, 293-T human kidney cells, A2870 human ovarian cells, A431 human epithelial cells, B35 rat neuroblastoma cells, BHK-21 hamster kidney cells, BR293 human breast cells, CHO Chinese hamster ovary cells, CORL23 human lung cells, HeLa cells, or Jurkat cells. The sample may comprise a homogenous or mixed population of microorganisms including one or more of viruses, bacteria, protists, prokaryotes, vesicular algae, archaea or fungi. The biological sample may be a urine specimen, a vaginal swab, a cervical swab, an anal swab or a cheek swab. The biological sample may be obtained from a hospital, laboratory, clinical or medical laboratory.

However, the devices and methods described herein are not limited to performing molecular diagnostic tests on human samples. In some embodiments, any of the devices and methods described herein can be used with veterinary, food, and/or environmental samples. Examples of environmental sources include, but are not limited to, farmlands, lakes, rivers, reservoirs, vents, walls, roofs, soil samples, plants, and swimming pools. Examples of industrial sources include, but are not limited to, clean rooms, hospitals, food processing areas, food production areas, food, medical laboratories, pharmacies, and drug dispensing centers. Examples of subjects from which polynucleotides may be isolated include multicellular organisms such as fish, amphibians, reptiles, birds, and mammals. Examples of mammals include primates (e.g., apes, monkeys, gorillas), rodents (e.g., mice, rats), cows, pigs, sheep, horses, dogs, cats, and rabbits. In some examples, the mammal is a human.

In some embodiments, any of the devices and methods described herein can include a sample buffer (e.g., within the sample preparation module, sample transport shunt, or reagent module) and/or can mix the sample buffer with the biological sample, or can use the sample buffer as a wash/blocking solution, as described herein. In some cases, the sample buffer may include bovine serum albumin and/or a detergent. In some cases, the sample buffer comprises about 0.1% to 5% bovine serum albumin. In some cases, the sample buffer comprises about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, or 5% bovine serum albumin. In some cases, the sample buffer comprises about 0.1% to 20% detergent. In some cases, the sample buffer comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% detergent. In some cases, the detergent is Tween-20. The choice of sample buffer to be used may depend on the intended method. For example, the choice of sample buffer may be different when a washing step is used than when a washing step is not used. If a washing step is not used, the sample buffer may be a buffer suitable for lysis and subsequent PCR reactions.

In some embodiments, the sample buffer may include Tris HCL, Tween-80, BSA, Proclin, and Antifoam SE-15. In some embodiments, the sample buffer may have the following composition: 50mM Tris pH8.4, Tween-80, 2% (w/v), BSA, 0.25% (w/v), Proclin 3000.03% (w/v) and Antifoam SE-15, 0.002% (v/v), purified water. Tris HCl is a common buffer for PCR. When it is heated during thermocycling, the pH may drop, for example, a Tris buffer at pH8.4 at a temperature of 25 ℃ may drop to a pH of about-7.4 when heated to about 95 ℃. The concentration may range from 0.1mM to 1M. The pH may range from 6 to 10. Any other PCR compatible buffer, such as HEPES, may be used. Proclin 300 is a broad spectrum antimicrobial agent that acts as a preservative to ensure long shelf life of the collection medium. It can be used in the range of 0.01% (w/v) to 0.1% (w/v). Many other antimicrobial agents are known in the art and may be used in the sample buffer. In some embodiments, the reagent or wash buffer may include Antifoam SE-15 to reduce foam formation during manufacture and fluid movement through the device. It can be used at 0.001% (v/v) to 1% (v/v). Any other antifoaming agent may also be used, for example, Antifoam 204, Antifoam A, Antifoam B, Antifoam C or Antifoam Y-30.

In some embodiments, any of the amplification modules described can be configured to perform "rapid" PCR (e.g., complete at least 30 cycles in less than about 10 minutes), and to rapidly generate an output signal (e.g., by a detection module). In other words, the amplification modules described herein can be configured to handle a variety of volumes, have a variety of dimensional sizes, and/or be constructed from materials that facilitate rapid PCR or amplification in less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, or any range therebetween as described herein.

In some embodiments, any of the detection modules described herein can include capture probes having any suitable structure or composition. Such capture probes may be, for example, any of single-stranded nucleic acids, antibodies, or binding proteins. In some embodiments, the capture probe has the following general structure (the DNA base sequences herein are examples only, and will vary depending on the target amplicon):

5 '-end-/5 AmMC6/TCTCGTAAAGGGCAGCCCGCAAG-3' -end.

In other embodiments, the capture probe may be modified to also contain a spacer molecule, according to which structure:

5' end-/5 AmMC6// iSpl8/TCTCGTAAAGGGCAGCCCGCAAG-3' end wherein/5 AmMC 6/is a 5' amino modifying group C6-Integrated DNA Technologies,/iSpl 8/is an Int spacer 18-Integrated DNA Technologies. In other embodiments, the capture probe may be modified to include only the desired DNA bases, according to this structure:

5 'end-TCTCGTAAAGGGCAGCCCGCAAG-3' end.

In other embodiments, the capture probe further comprises additional non-target bases according to this structure:

5 'end-GGGGGGG TCTCGTAAAGGGCAGCCCGCAAG-3' end.

In some embodiments, the capture probe may be formulated, designed or engineered to have a relatively high melting temperature (Tm) value (e.g., about 67 ℃). In other embodiments, the capture probe may have a melting temperature (Tm) value in the following range: 35-85 ℃, 60-75 ℃, 65-70 ℃ or 66-68 ℃. One advantage of a capture probe with a high Tm value is that the flow cell can be heated to a wide temperature range during operation without causing the capture probe to release the target amplicon.

In some embodiments, the capture probes are designed against sequences from Neisseria gonorrhoeae (Neisseria gonorrhoeae), chlamydia trachomatis, trichomonas vaginalis, Neisseria subflava (Neisseria subflava) and negative control sequences such as sequences from Bacillus atrophaeus (Bacillus atrophaeus) or random bases.

Some embodiments described herein relate to a computer storage product (which may also be referred to as a non-transitory processor-readable medium) with a non-transitory computer-readable medium having instructions or computer code thereon for performing various computer-implemented operations. The computer-readable medium (or processor-readable medium) is non-transitory in the sense that it does not include signals that are transitory in nature (e.g., propagating electromagnetic waves carrying information over a transmission medium such as space or cable). The media and computer code (also can be referred to as code) may be those designed and constructed for one or more specific uses. Examples of non-transitory computer readable media include, but are not limited to, magnetic storage media such as hard disks, floppy disks, and magnetic tape; optical storage media such as compact discs/digital video discs (CD/DVD), compact disc read-only memories (CD-ROM), and holographic devices; magneto-optical storage media such as optical disks; a carrier signal processing module; and hardware devices that are dedicated to storing and executing program code, such as Application Specific Integrated Circuits (ASICs), Programmable Logic Devices (PLDs), Read Only Memory (ROM), and Random Access Memory (RAM) devices.

Examples of computer code include, but are not limited to, microcode or microinstructions, machine instructions, such as those generated by a compiler, code for generating a web service, and files containing higher-level instructions that are executed by a computer using an interpreter. For example, embodiments may be implemented using an imperative programming language (e.g., C language, Fortran language, etc.), a functional programming language (Haskell language, Erlang language, etc.), a logical programming language (e.g., Prolog language), an object-oriented programming language (e.g., Java language, C + + language, etc.), or other suitable programming languages and/or development tools. Other examples of computer code include, but are not limited to, control signals, encrypted code, and compressed code.

The processor included within the control module (as well as any of the processors and/or controllers described herein) may be any processor configured to, for example, write data to and read data from the memory of the controller, and execute instructions/methods stored within the memory. Further, the processor may be configured to control the operation of other modules within the controller (e.g., the temperature feedback module and the flow module). In particular, the processor may receive signals including temperature data, current measurements, and the like and determine the amount of power and/or current supplied to each heater assembly, the desired timing and sequence of piston pulses, and the like. For example, in some embodiments, the controller may be an 8-bit PIC microcontroller that will control the power delivered to the various heating assemblies and components within the amplification module 4600. The microcontroller may also contain code for and/or be configured to minimize instantaneous power requirements on the power supply.

In other embodiments, any of the processors described herein may be, for example, an Application Specific Integrated Circuit (ASIC) or a combination of ASICs designed to perform one or more dedicated functions. In still other embodiments, the microprocessor may be an analog or digital circuit, or a combination of circuits.

Any of the memory devices described herein may be any suitable device, such as, for example, a read-only memory (ROM) component, a Random Access Memory (RAM) component, an electronically programmable read-only memory (EPROM), an erasable electronically programmable read-only memory (EEPROM), a register, a cache memory, and/or a flash memory. Either module (pressure feedback module and position feedback module) may be implemented by a processor and/or stored in memory.

Although various embodiments have been described as having particular combinations of features and/or components, other embodiments are possible having any combination of features and/or components from any of the embodiments described above.

Any of the devices and methods described herein can be used to detect the presence or absence of nucleic acids associated with one or more bacterial cells in a biological sample. In some embodiments, the one or more bacterial cells are pathogens. In some embodiments, the one or more bacterial cells are infectious. Non-limiting examples of bacterial pathogens that can be detected include Mycobacteria (mycobacterium) (e.g., mycobacterium tuberculosis (m.tuberculosis), mycobacterium bovis (m.bovis), mycobacterium avium (m.avium), mycobacterium leprae (m.leprae), and mycobacterium africanum (m.africanum)), rickettsia, mycoplasma, chlamydia, and legionella. Some examples of bacterial infections include, but are not limited to, infections caused by: gram-positive bacteria (Bacillus) (e.g. Listeria (Listeria), bacilli (Bacillus) such as Bacillus anthracis (Bacillus ankhraris), Erysipelothrix (Erysipelothrix) species), gram-negative bacteria (e.g. Bartonella (Bartonella), Brucella (Brucella), Campylobacter (Campylobacter), Enterobacter (Enterobacter), Escherichia (Escherichia), Francisella (Francisella), haemophilus (Hemophilus), Klebsiella (Klebsiella), Morganella (Morganella), Proteus (Proteus), Providencia (Providencia), Pseudomonas (Pseudomonas), Salmonella (Salmonella), Serratia (serrtia), shirtia (Shigella), Shigella (vibrtia), and yersinia (yersinia), including Borrelia species (Borrelia), including Borrelia (Borrelia), and Borrelia (Borrelia), including Borrelia (Borrelia) species, Borrelia (Borrelia), Borrelia (Borrelia), and Borrelia (Borrelia) species, including Borrelia (Borrelia) species, e (Borrelia, e, Borrelia, and bacterial species including Borrelia (Borrelia) species, e (Borrelia, Anaerobes (e.g., Actinomyces and Clostridium species), gram-positive and negative cocci, Enterococcus (Enterococcus) species, Streptococcus (Streptococcus) species, Pneumococcus (pneumcoccus) species, Staphylococcus (Staphylococcus) species, and Neisseria (Neisseria) species. Specific examples of infectious bacteria include, but are not limited to: helicobacter pylori (Helicobacter pylori), legionella pneumophila), Mycobacterium tuberculosis, Mycobacterium avium, intracellular Mycobacterium (Mycobacterium intracellularis), Mycobacterium kansasii (Mycobacterium kansaii), Mycobacterium gordonii (Mycobacterium gordonae), Staphylococcus aureus (Staphylococcus aureus), Neisseria gonorrhoeae (Neisseria gordoniae), Neisseria meningitidis (Neisseria meningitidis), Listeria monocytogenes (Listeria monocytogenes), Streptococcus pyogenes (Streptococcus pyelogenes) (group A Streptococcus), Streptococcus agalactiae (Streptococcus agalactiae) (group B Streptococcus), Streptococcus viridis (Streptococcus faecalis), Streptococcus faecalis (Streptococcus faecalis), Streptococcus faecal (Streptococcus faecalis), Streptococcus pneumoniae (Streptococcus faecalis), Streptococcus faecalis (Streptococcus faecalis), Mycobacterium kansuensis (Streptococcus pneumoniae), Mycobacterium kansuis (Streptococcus pneumoniae), Mycobacterium kansasanberella (Streptococcus pneumoniae), Mycobacterium tuberculosis (Streptococcus pneumoniae (Streptococcus), Mycobacterium tuberculosis (Streptococcus pneumoniae), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae (Streptococcus), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus pneumoniae (Streptococcus), Streptococcus pneumoniae (Streptococcus pneumoniae), Streptococcus (Streptococcus), Streptococcus pneumoniae), Streptococcus (Streptococcus) Bacillus coli (Streptococcus), Klebsiella pneumoniae (Klebsiella pneumoniae), Pasteurella multocida (Pasteurella multocida), Fusobacter nucleatum (Fusobacterium nucleatum), Streptococcus candidus (Streptomyces moniliformis), Treponema pallidum (Treponema pallidum), Treponema pallidum (Treponema pertenue), Leptospira (Leptospira), Rickettsia (Rickettsia) and Actinomyces (Actinomyces israelii), Acinetobacter (Acinetobacter), Bacillus (Bacillus), Bordetella (Bordetella), Borrelia (Borrelia), Brucella (Brucella), Campylobacter (Campylobacter), Chlamylacter (Campylobacter), Chlamydia (Chlamydia), Clostridium (Clostridium), Clostridium (Corynebacterium), Clostridium (Corynebacterium), Clostridium (Corynebacterium) and Escherichia (Corynebacterium) are used in (Corynebacterium, Escherichia (Corynebacterium), Escherichia (Corynebacterium), Escherichia (Corynebacterium), Escherichia (Corynebacterium) and Escherichia), Escherichia (Corynebacterium) are (Corynebacterium) and Escherichia), Escherichia (Corynebacterium) are (Corynebacterium) are (, Bordetella pertussis (Bordetella pertussis), Brucella abortus (Brucella abortus), Brucella canis (Brucella canis), Brucella ovis (Brucella melitensis), Brucella suis (Brucella suis), Campylobacter jejuni (Campylobacter jejuni), Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci (Chlamydophila psittaci), Clostridium botulinum (Clostridium botulinum bortetulinum), Clostridium difficile (Clostridium difficile), Clostridium perfringens (Clostridium perfringens), Corynebacterium diphtheriae (Corynebacterium diphenii), Enterobacter sakawakazae (Enterobacter faecalis), Enterobacter faecalis (Enterobacter faecalis), Escherichia coli (Enterobacter faecalis), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium tuberculosis (Leccinellicola), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis (Mycobacterium) and Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis (Mycobacterium), Mycobacterium tuberculosis, Mycobacterium ulcerosa (Mycobacterium ulcerons), Mycoplasma pneumoniae (Mycoplasma pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Rickettsia rickettsii (Rickettsia rickettsii), Salmonella typhi (Salmonella typhi), Salmonella typhimurium (Salmonella typhimurium), Salmonella enterica (Salmonella enterica), Shigella sonnei (Shigella sonnei), Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus saprophyticus (Staphylococcus saphenocarpicus), Shigella maltophilia (Stenotrophora), Vibrio cholerae (Vibrio cholerae), Yersinia pestis (Yersinia pestis), and the like. In some cases, the infectious bacterium is neisseria gonorrhoeae or chlamydia trachomatis.

Any of the devices and methods described herein can be used to detect the presence or absence of nucleic acids associated with one or more viruses in a biological sample. Non-limiting examples of viruses include herpes viruses (e.g., Human Cytomegalovirus (HCMV), herpes simplex virus I (HSV-1), herpes simplex virus 2(HSV-2), Varicella Zoster Virus (VZV), epstein-barr virus), influenza a and Hepatitis C Virus (HCV) or picornaviruses such as coxsackievirus B3(CVB 3). Other viruses may include, but are not limited to, hepatitis B virus, HIV, poxviruses, hepadnaviruses (hepadavirus), retroviruses, and RNA viruses such as flavivirus, togavirus, coronavirus, hepatitis delta virus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus, adenovirus, human herpesvirus, type 8, human papilloma virus, BK virus, JC virus, smallpox, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, norwalk virus, coxsackievirus, hepatitis a virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis c virus, yellow fever virus, dengue virus, west nile virus, rubella virus, hepatitis e virus, and Human Immunodeficiency Virus (HIV). In some embodiments, the virus is an enveloped virus. Examples of such enveloped viruses include, but are not limited to, viruses that are members of the families: hepadnaviridae, herpesviridae, iridoviridae, poxviridae, flaviviridae, togaviridae, retroviridae, coronaviridae, filoviridae, rhabdoviridae, bunyaviridae, orthomyxoviridae, paramyxoviridae, and arenaviridae. Other examples include the use of a combination of,but are not limited to, hepadnavirus B hepatitis virus (HBV), woodchuck hepatitis virus, geotrichia hepatitis (hepadnaviridae), duck hepatitis B virus, herou hepatitis B virus, Herpes Simplex Virus (HSV) types 1 and 2, varicella zoster virus, Cytomegalovirus (CMV), Human Cytomegalovirus (HCMV), Mouse Cytomegalovirus (MCMV), Guinea Pig Cytomegalovirus (GPCMV), Epstein Barr Virus (EBV), human herpesvirus 6(HHV variants a and B), human herpesvirus 7(HHV-7), human herpesvirus 8(HHV-8), kaposi's sarcoma-associated herpesvirus (hv), poxvirus B vaccinia virus, smallpox virus (variola virus), smallpox virus (smallpox virus), vaccinia virus, camelpox virus, mouse podovirus (ectoria virus), mouse poxvirus virus, murine poxvirus (hepadnavirus), yavirus B, herpes virus B (, Leporipoxviruses, raccoon poxviruses, molluscum contagiosum viruses, aphtha viruses, milking-birch nodaviruses, bowen-billetto virus (papula-contagiosum virus), sheep poxviruses, goat poxviruses, sarcoid dermatosis viruses, fowl pox viruses, canary pox viruses, pigeon poxviruses, myxoma viruses, rabbit fibroma viruses, squirrel fibroma viruses, pig pox viruses, tenna river pox viruses, yapoxvirus, flavivirus dengue viruses, Hepatitis C Viruses (HCV), GB hepatitis viruses (GBV-A, GBV-B and GBV-C), West Nile viruses, yellow fever viruses, St.Louis encephalitis viruses, Japanese encephalitis viruses, Powassan viruses (Powassan viruses), tick-borne encephalitis viruses (tick-borne enceisis viruses), Kosanual Forest disease viruses (Kyasanur Forest disease virus), Togaviruses (Togavirus), Venezuelan Equine Encephalitis (VEE) virus, chikungunya virus (chikungunya virus), ross river virus, mairo virus, sindbis virus, rubella virus, retrovirus Human Immunodeficiency Virus (HIV) types 1 and 2, human T cell leukemia virus (HTLV) types 1, 2 and 5, Mouse Mammary Tumor Virus (MMTV), Rous Sarcoma Virus (RSV), lentiviruses, coronaviruses, Severe Acute Respiratory Syndrome (SARS) virus, filovirus ebola virus, marburg virus, Metapneumovirus (MPV) such as Human Metapneumovirus (HMPV), rabies virus, vesicular stomatitis virus, bunyavirus,Crimean-congo hemorrhagic fever virus, rift valley fever virus, lacrosse virus, hantavirus, orthomyxovirus, influenza virus (type a, type B, type c), paramyxovirus, parainfluenza virus (PIV1, types 2 and 3), respiratory syncytial virus (types a and B), measles virus, mumps virus, arenavirus, lymphocytic choriomeningitis virus, junin virus, marulo virus, melon nreto virus, lassa virus, Ampari virus, friexovirus, epsiper virus, mobarala virus, mopeya virus, lothino virus, barana virus, picard virus, Punta Torn Virus (PTV), tacaribe virus and tera virus. In some embodiments, the virus is a non-enveloped virus, examples of which include, but are not limited to, viruses that are members of the following families: parvoviridae, circoviridae, polyomaviridae, papilloma viridae, adenoviridae, iridoviridae, reoviridae, birnaviridae, sepaviridae and picornaviridae. Specific examples include, but are not limited to, canine parvovirus, parvovirus Bl9, porcine circovirus type 1 and 2, BFDV (Rheukoid disease Virus, Chicken anemia Virus, polyoma Virus, Simian Virus 40(SV40), JC Virus, BK Virus, Homopittus Alopecurus Virus, human papilloma Virus, Bovine Papilloma Virus (BPV) type 1, Google Rabbit papilloma Virus, human adenoviruses (HAdV-A, HAdV-B, HAdV-C, HAdV-D, HAdV-E and HAdV-F), avian adenovirus A, bovine adenovirus D, frog adenovirus, reovirus, human circovirus, human colti Virus, mammalian proenterovirus, Bluetongue Virus, Rotavirus A, Rotaviruses (groups B through G), Colorado tick fever Virus, aquatic reovirus A, polyhedrosis Virus 1, Fizea Virus, rice dwarf Virus, Rice ragged dwarf virus, insect non-inclusion body virus 1, fungal reovirus 1, birnavirus, bursal disease virus, pancreatic necrosis virus, calicivirus, porcine vesicular ulcer virus, rabbit hemorrhagic disease virus, norwalk virus, Sapovirus, picornavirus, human poliovirus (1-3), human saxivirus Al-22, 24(CAl-22 and CA24, CA23 (Eicovirus 9)), human saxivirus (Bl-6(CBl-6)), human echovirus 1-7,9,11-27,29-33, viluishViruses, simian enteroviruses 1-18(SEVI-18), porcine enteroviruses 1-11(PEVl-11), bovine enteroviruses 1-2(BEVI-2), hepatitis A, rhinoviruses, hepatotrophic viruses, cardioviruses, foot and mouth disease viruses, and echoviruses. The virus may be a bacteriophage. Examples of bacteriophages include, but are not limited to, T4, TS, lambda, T7, G4, Pl,Thermophilus viruses 1, M13, MS2, Q β,X174, Φ 29, PZA, Φ 15, BS32, Bl03, M2Y (M2), Nf, GA-I, FWLBcl, FWLBc2, FWLLm3, B4. The reference database may include sequences of phage that are pathogenic, protective, or both. In some cases, the virus is selected from a member of the families: flaviviridae (e.g., members of the flavivirus, pestivirus, and hepatitis c virus (Hepacivirus) genera) including hepatitis c virus, yellow fever virus; tick-borne viruses such as Gade-Grave Valley virus, Kadan-M virus, Kosaronary forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassen virus, Royal farm virus, Kaxi virus, tick-borne encephalitis virus, Newdorff virus, Softragi virus, sheep jump virus and Nagi Schw virus; seabird tick-borne viruses such as rice spot virus, somali ziriff virus and kanehir virus; mosquito-borne viruses such as an arnia virus, a dengue virus, a cathodo virus, a caspaselli virus, a kotango virus, a japanese encephalitis virus, a murray valley encephalitis virus, a st louis encephalitis virus, a soyasu soil virus, a west nile virus, an yawendt virus, a koeba virus, a bagazar virus, an ire virus (ileus virus), an israel-turkey meningoencephalitis virus, an entaya virus, a tabsu virus, a zakhaki virus, a bobobo virus, an egjyas virus, a vermilion gla virus, a sabo virus, a seebeck virus, a uda S virus, a wessel braun virus, a yellow fever virus; and viruses with unknown arthropod vectors, such as Entber batViruses, tassel viruses, apolexvirus, hiyaviruses, huoshan virus, hutuyapapa virus, modus operandi virus, salbijous virus, saint palita virus, bocarva bat virus, caritavirus, dacha bat virus, mengtana bat leukoencephalitis virus, pink campsis bat virus, ricjo bravo virus, tacan bat virus and cell fusion agent viruses. In some cases, the virus is selected from a member of the families: arenaviridae, which includes epstein-barr virus, lassa virus (e.g., strain Josiah, LP, or GA 391), lymphocytic choriomeningitis virus (LCMV), mobara virus, mopeya virus, amapali virus, friexole virus, melon nritto virus, junin virus, ratino virus, maculo virus, orihuas virus, parnam virus, piceide virus, pirimid virus, sapica virus, tacarib virus, tera virus, whitewater river virus, Chapare virus, and Lujo virus. In some cases, the virus is selected from a member of the families: bunyaviridae, (e.g., members of the hantavirus genus, nelovirus genus, orthomyxovirus genus, and phlebovirus genus) which include hantaviruses, sinovirus, dougby virus, bunyaviras, rift valley fever virus, larksos virus, pomtorola virus (PTV), california encephalitis virus, and crimia-congo hemorrhagic fever (CCHF) virus. In some cases, the virus is selected from a member of the families: filoviridae, which includes ebola viruses (e.g., zaire, sudan, ivory coast, reston, and uda strains) and marburg viruses (e.g., angora, Ci67, mosoks, poppy, livin, and victoria strains); members of the togaviridae family (e.g., members of the alphavirus genus) including venezuelan equine encephalitis Virus (VEE), eastern equine encephalitis virus (EEE), western equine encephalitis virus (WEE), sindbis virus, rubella virus, siemens forest virus, ross river virus, bam forest virus, o-neviras, and chikungunya virus; members of the poxviridae (e.g., members of the orthopoxvirus genus) that include variola virus, monkeypox virus, and vaccinia virus; members of the herpes virus family, including herpes simplex virus (HSV; types 1, 2 and 6), human herpes virus (e.g. HSV)Types 7 and 8), Cytomegalovirus (CMV), epstein-barr virus (EBV), varicella-zoster virus and kaposi sarcoma-associated herpes virus (KSHV); members of the orthomyxoviridae family, which include influenza viruses (type a, b, and c), such as H5Nl avian influenza virus or HINI swine influenza virus; members of the family coronaviridae, including the Severe Acute Respiratory Syndrome (SARS) virus; members of the rhabdoviridae family, which includes rabies virus and Vesicular Stomatitis Virus (VSV); members of the paramyxoviridae family, which include human Respiratory Syncytial Virus (RSV), newcastle disease virus, hendra virus, nipah virus, measles virus, rinderpest virus, canine distemper virus, sendai virus, human parainfluenza viruses (e.g., 1, 2, 3, and 4), rhinoviruses, and mumps virus; members of the picornaviridae family, which include poliovirus, human enterovirus (A, B, C and D), hepatitis a virus, and coxsackievirus; members of the hepadnaviridae family, which includes hepatitis b virus; members of the papillomavirus family, which include human papillomaviruses; members of the parvoviridae family, which include adeno-associated viruses; members of the astroviridae family, which include astroviruses; members of the polyomaviridae family, which include JC virus, BK virus and SV40 virus; members of the family caliciviridae, which include norwalk virus; members of the reoviridae family, which include rotaviruses; and members of the Retroviridae family, which include human immunodeficiency viruses (HIV; e.g., types I and 2), and human T-lymphoblastoviruses types I and II (HTLV-1 and HTLV-2, respectively).

Any of the devices and methods described herein can be utilized to detect the presence or absence of nucleic acids associated with one or more fungi in a biological sample. Examples of infectious fungal pathogens include, without limitation, at least three genera of Aspergillus (Aspergillus), Blastomyces (Blastomyces), Coccidioides (Coccidioides), Cryptococcus (Cryptococcus), Histoplasma (Histoplasma), Paracoccidioides (Paracoccidiales), Sporothrix (Sporothrix), and Zygomycetes (Zygomycetes). The above fungi, as well as many others, can cause disease in pets and companion animals. The teachings of the present invention include a substrate that is in direct or indirect contact with an animal. Organisms causing animal diseases include Malassezia furfur (Malassezia furur), Epidermophyton floccosum (epimophyton floccosur), Trichophyton mentagrophytes (trichophytons), Trichophyton rubrum (Trichophyton rubrum), Trichophyton brevicum (Trichophyton tonsurans), Trichophyton equisetum (Trichophyton quinum), dermatophyton conoides (dermatophyllum comosum), Microsporum canis (Microsporum canis), Microsporum ovani (microsporubii), Microsporum gypseum (Microsporum gypseum), pityrosporum ovale (Malassezia ovata), pseudomonas pseudoeherulea (pseudomonas aeruginosa), scoparia (scoparia), actinomyces sporum (sclerotium), and Candida albicans (Candida albicans). Further examples of fungal infection pathogens include, but are not limited to, Aspergillus, Blastomyces dermatitidis (Blastomyces dermatitidis), Candida, Coccidioides immitis, Cryptococcus neoformans (Cryptococcus neoformans), Histoplasma capsulatum var. capsulatum (Histoplasma capsulatum var. capsulatum), Brazilian Blastomyces (Paracoccus brasiliensis), Sporothrix schenckii (Sporothrix schenckii), Zygocetes p, Absidia umbellata (Absidia corembifera), Rhizomucor miens (Rhizomucor pusillus) or Rhizopus arrhizus (Rhizopus arrhizus).

Any of the devices and methods described herein can be used to detect the presence or absence of nucleic acids associated with one or more parasites in a biological sample. Non-limiting examples of parasites include Plasmodium (Plasmodium), Leishmania (Leishmania), Babesia (Babesia), Treponema (Treponema), Borrelia (Borrelia), Trypanosoma (Trypanosoma), Toxoplasma (Toxoplasma gondii), Plasmodium falciparum (Plasmodium falciparum), Plasmodium vivax (p.vivax), Plasmodium ovale (p.ovale), Plasmodium malariae (p.malariae), Trypanosoma (Trypanosoma spp.) or Legionella spp. In some cases, the parasite is Trichomonas vaginalis.

Claims (80)

1. A method of detecting nucleic acids using a molecular diagnostic test device, the method comprising:

coupling the molecular diagnostic test device to a power source;

transferring a biological sample through an input opening into a sample preparation module within the molecular diagnostic test device;

actuating the molecular diagnostic test device with only a single action to cause the molecular diagnostic test device to:

A) heating the biological sample by a heater of the sample preparation module to lyse a portion of the biological sample to produce an input sample;

B) transferring the input sample to an amplification module within the molecular diagnostic test device, the amplification module defining a reaction volume;

C) heating the input sample within the reaction volume to amplify the nucleic acids within the input sample, thereby generating an output solution containing target amplicons; and

D) within a detection module within the molecular diagnostic test device, reacting each of: (i) the output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution, the detection module comprising a detection surface configured to capture the target amplicon to produce the signal; and

the result associated with the signal is read.

2. The method of claim 1, wherein the single action is capping the molecular diagnostic test device.

3. A method of detecting nucleic acids using a molecular diagnostic test device, the method comprising:

coupling the molecular diagnostic test device to a power source;

transferring a biological sample through an input opening into a sample preparation module within the molecular diagnostic test device;

covering said input opening with a cover connected to said molecular diagnostic test device;

causing the molecular diagnostic test device to:

A) heating the biological sample by a heater of the sample preparation module to lyse a portion of the biological sample to produce an input sample;

B) transferring the input sample to an amplification module within the molecular diagnostic test device, the amplification module defining a reaction volume;

C) heating the input sample within the reaction volume to amplify the nucleic acids within the input sample, thereby generating an output solution comprising target amplicons; and

D) within a detection module within the molecular diagnostic test device, reacting each of: (i) the output solution and (ii) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution, the detection module comprising a detection surface configured to capture the target amplicon to produce the signal; and

the result associated with the signal is read.

4. The method of claim 3, wherein the molecular testing device is a single use device, the method further comprising:

discarding the molecular testing device after the readout.

5. The method of claim 3, wherein coupling the molecular diagnostic test device to the power source comprises any one of: coupling the molecular diagnostic test device to the power source through a wire, coupling a terminal of the power source to a corresponding terminal of the molecular diagnostic test device, or removing an insulating member between the power source and an electronic control within the molecular diagnostic test device.

6. The method of claim 3, wherein said covering the input opening is performed by a single action.

7. The method of claim 6, wherein the covering the input opening comprises: A) moving the sealing portion of the lid over the input opening, B) actuating a switch to provide power from the power source to the heater, and C) releasing the reagent from a sealed reagent container within the molecular diagnostic test device by the single action.

8. The method of claim 6, wherein after the covering, a latch portion of the cap irreversibly engages the molecular diagnostic test device to prevent the input opening from being opened.

9. The method of claim 7, the method further comprising:

storing said molecular diagnostic test device comprising said sealed reagent container for at least 6 months prior to said coupling.

10. A method of detecting a nucleic acid, the method comprising:

mixing a reverse transcriptase with a biological sample within a sample preparation module to form a reverse transcription solution;

heating, within the sample preparation module, the reverse transcription solution to a first temperature within a lysis temperature range to release ribonucleic acid (RNA) molecules;

heating, within the sample preparation module, the reverse transcription solution to a second temperature within a reverse transcription temperature range to produce complementary deoxyribonucleic acid (cDNA) molecules;

heating the reverse transcription solution to a third temperature above an inactivation temperature within the sample preparation module to cause inactivation of the reverse transcriptase; and;

the reverse transcription solution is transferred to an amplification module.

11. The method of claim 10, wherein the reverse transcription solution is free of ribonuclease inhibitors.

12. The method of claim 11, wherein the heating to the first temperature and the heating to the second temperature are performed in at least 10 minutes.

13. The method of claim 12, wherein:

the sample preparation module includes a flow member defining a flow path and a heater coupled to the flow member; and

the heating to a first temperature and the heating to a second temperature are performed by actuating the heater and conveying the reverse transcription solution through the flow path.

14. The method of claim 13, wherein the flow path is a serpentine flow path.

15. The method of claim 12, wherein the volume of the reverse transcription solution is at least 50 microliters.

16. The method of claim 12, wherein:

the heating to the first temperature comprises heating the reverse transcription solution at a rate of 0.1 degrees Celsius per second to 100 degrees Celsius per second; and

the heating to the second temperature comprises heating the reverse transcription solution at a rate of 0.1 degrees Celsius per second to 100 degrees Celsius per second.

17. The method of claim 12, wherein the biological sample is an unfiltered sample.

18. The method of claim 13, wherein the flow rate of the reverse transcription solution is such that the reverse transcription solution is heated to the third temperature for a time of at least about 25 seconds, the third temperature being from about 92 degrees celsius to about 98 degrees celsius.

19. An apparatus, the apparatus comprising:

a housing;

a sample preparation module within the housing, the sample preparation module defining a sample input volume to receive a biological sample and an input opening through which the sample input volume is accessible, the sample preparation module comprising a heater configured to heat the biological sample to produce an input solution;

a reagent module disposed within the housing, the reagent module comprising a reagent container containing a detection reagent configured to facilitate generation of a signal indicative of the presence of a target amplicon from the input solution, the detection reagent sealed within the reagent container;

a detection module comprising a detection surface configured to capture the target amplicons from the input solution, the detection module in fluid communication with the reagent module such that the signal is generated in response to the reagent delivered into the detection module; and

a lid movably connected with the housing, the lid including a sealing portion, a switching portion, and a reagent actuator, the lid configured to move relative to the housing between a first lid position and a second lid position, the input opening exposed when the lid is in the first lid position, the sealing portion of the lid covering the input opening when the lid is in the second lid position, the switching portion configured to actuate a switch to provide power to the heater when the lid is moved from the first lid position to the second lid position, the reagent actuator configured to release the reagent from the sealed reagent container when the lid is moved from the first lid position to the second lid position.

20. The apparatus of claim 19, wherein the lid comprises a detent that irreversibly engages at least one of the housing, the sample preparation module, or the reagent module to hold the lid at the second lid position.

21. The apparatus of claim 20, wherein the detent of the cover is a protrusion that is matingly received within an opening defined by the housing when the cover is in the second cover position.

22. The apparatus of claim 19, wherein:

the input opening of the housing is a first input opening;

the cover defines a second input opening that is flush with the first input opening when the cover is in the first cover position.

23. The apparatus of claim 22, wherein the seal of the lid fluidly isolates the sample input volume when the lid is in the second lid position.

24. The apparatus of claim 19, wherein:

the reagent module comprises a reagent housing defining a reagent reservoir into which the reagent is released from the sealed reagent container when the piercer pierces a portion of the reagent container, the reagent reservoir configured to be placed in fluid communication with the detection module; and

the reagent actuator includes a protrusion that applies a force to cause the piercer to pierce the portion of the reagent container when the lid is moved from the first lid position to the second lid position.

25. The apparatus of claim 24, wherein:

the reagent actuator of the lid is a first reagent actuator; and

the reagent module includes a second reagent actuator between the lid and the reagent housing, the second reagent actuator including a deformable member, the protrusion of the first reagent actuator configured to apply a force to the deformable member such that the deformable member moves, thereby transferring the force to move the reagent container within the reagent reservoir and into contact with the piercer.

26. The apparatus of claim 24, wherein:

the reagent module comprising a reagent container support member configured to connect the reagent container within the reagent reservoir, the reagent container support member having a sealing portion connected to the reagent housing to fluidly isolate the reagent reservoir and a connecting portion connected to a portion of the reagent container to support the reagent container in a first container position within the reagent reservoir, the reagent container being distal from the piercer when the reagent container is in the first container position,

the reagent container support member is configured to deform from an initial configuration to a deformed configuration in response to a force applied by the reagent actuator, the reagent container moving to a second container position within the reagent reservoir and in contact with the piercer when the reagent container support member transitions from the initial configuration to the deformed configuration.

27. The apparatus of claim 24, wherein the reagent container support member is biased in the initial configuration.

28. The apparatus of claim 19, the apparatus further comprising:

an amplification module within the housing, the amplification module configured to receive the input solution from the sample preparation module, the amplification module configured to heat the input solution to amplify nucleic acids within the input solution, thereby generating a detection solution comprising the target amplicons.

29. The apparatus of claim 28, the apparatus further comprising:

a fluid pump disposed within the housing, the fluid pump configured to generate an input flow of the input solution within the amplification module; and

a control module within the housing, the control module comprising the switch and a processor, the switch providing power to the processor when the lid is moved from the first lid position to the second lid position, the processor configured to adjust a power input to the fluid pump to control a rate of the input flow of the sample within the amplification module.

30. The apparatus of claim 19, the apparatus further comprising:

a control module within the housing, the control module including the switch and a processor configured to control delivery of power to the heater, the switch portion of the lid including a switch protrusion configured to engage with the switch when the lid is in the second lid position.

31. The apparatus of claim 19, wherein the reagent is one of a first reagent configured to bind to the target amplicon in response to the first reagent being delivered into the detection module or a second reagent configured to generate the signal when catalyzed by the first reagent.

32. The apparatus of claim 31, wherein the second reagent is a precipitation substrate formulated to produce insoluble colored particles when the second reagent is contacted with the first reagent.

33. The apparatus of claim 19, wherein:

the reagent container is a first reagent container;

the reagent is a first reagent that is one of a catalytic reagent formulated to bind to the target amplicon in response to the first reagent being delivered into the detection module or a precipitating reagent formulated to produce the signal when catalyzed by the catalytic reagent; and

the reagent module includes a second reagent container containing a solution including a wash buffer and a blocking buffer configured to reduce attachment of the target amplicons within the detection module away from the detection surface.

34. A method of detecting nucleic acids using a molecular diagnostic test device, the method comprising:

transferring a first volume of a first reagent solution from a reagent module within the molecular diagnostic test device into a detection module within the molecular diagnostic test device at a first time, the detection module comprising a detection surface configured to capture a target amplicon associated with the nucleic acid, the first reagent solution comprising a blocking agent and a wash buffer, the first volume of the first reagent solution comprising an amount of the blocking solution sufficient to adsorb to a surface within the detection module;

transferring a sample solution comprising the target amplicon into the detection module at a second time such that the target amplicon is captured on the detection surface;

transferring a second reagent solution into the detection module after the second time, the second reagent solution being formulated to cause generation of a signal indicative of the presence of the target amplicon within the sample solution; and

transferring a second volume of the first reagent solution into the detection module after the second time, the second volume of the first reagent solution comprising the wash buffer in an amount sufficient to remove unbound components from at least one of the sample solution or the second reagent solution from the detection module.

35. The method of claim 34, wherein the molecular diagnostic test device is a stand-alone molecular diagnostic test device and the detection method is performed without any external instrumentation.

36. The method of claim 34, wherein the reagent module includes a first reagent container within which the first reagent solution is sealed prior to the first time transfer, the method further comprising:

releasing the first reagent solution from the first reagent container within the molecular diagnostic test device.

37. The method of claim 36, the method further comprising:

storing said molecular diagnostic test device comprising said sealed first reagent container for at least 6 months prior to said releasing.

38. The method of claim 36, wherein:

the reagent module includes a reagent housing defining a reagent reservoir;

said releasing a first reagent solution comprises releasing said first reagent solution into said reagent reservoir;

transferring a first volume of the first reagent solution at the first time comprises transferring a first volume of the first reagent solution from the reagent reservoir into the detection module and returning at least a portion of the first volume of the first reagent solution from the detection module to the reagent reservoir; and

delivering a second volume of the first reagent solution after the second time includes delivering the second volume of the first reagent solution from the reagent reservoir, the second volume including the first volume returned to the portion of the reagent reservoir.

39. The method of claim 38, wherein:

delivering a first volume of the first reagent solution at the first time, delivering the sample solution at the second time, and delivering a second volume of the first reagent solution after the second time are each performed by a fluid pump within the housing.

40. The method of claim 36, wherein the blocking agent comprises bovine serum albumin and the wash buffer comprises a detergent.

41. The method of claim 40, wherein the first reagent solution comprises 0.02% -5% bovine serum albumin and 0.05% -10% detergent.

42. The method of claim 34, the method further comprising:

transferring an input sample to an amplification module within the molecular diagnostic test device prior to the second time, the amplification module defining a reaction volume; and

heating the input sample within the reaction volume to amplify the nucleic acids within the input sample, thereby generating the sample solution comprising the target amplicon.

43. A method of detecting nucleic acids using a molecular diagnostic test device, the method comprising:

transferring a biological sample through an input opening into a sample preparation module within the molecular diagnostic test device;

actuating the molecular diagnostic test device to cause the molecular diagnostic test device to:

A) transferring a first volume of a reagent solution from a reagent module within the molecular diagnostic test device to a detection module within the molecular diagnostic test device, the detection module comprising a detection surface configured to capture a target amplicon associated with the nucleic acid, the reagent solution comprising a blocking agent and a wash buffer, the blocking agent being configured to adsorb to a surface within the detection module;

B) transferring the first volume of the reagent solution from the detection module back to the reagent module;

C) generating an output solution comprising the target amplicon associated with the nucleic acid from the biological sample;

D) passing the output solution into the detection module such that the target amplicons are captured on the detection surface; and

E) transferring a second volume of the reagent solution from the reagent module into the detection module to remove unbound components from the output solution from the detection module; and

reading out results associated with the target amplicons captured on the detection surface.

44. The method of claim 43, wherein the molecular detection device is a single-use device, the method further comprising:

discarding the molecular testing device after the readout.

45. The method of claim 43, wherein actuating the molecular diagnostic test device is performed by only a single action.

46. The method of claim 45, wherein the single action is closing a lid of the molecular diagnostic test device to retain the biological sample in the sample preparation module.

47. The method of claim 43, wherein:

the reagent module comprises a reagent container and a reagent housing defining a reagent reservoir within which the reagent solution is sealed prior to the actuation; and

actuating the molecular diagnostic test device further causes the molecular diagnostic test device to release the reagent solution into the reagent reservoir before the first volume of the reagent solution is transferred to the detection module.

48. A method of detecting a target RNA molecule using a disposable molecular diagnostic test device, the method comprising:

transferring an input sample to a reverse transcription module within a housing of the disposable molecular diagnostic test device;

heating the input sample within the reverse transcription module to produce target DNA molecules associated with the target RNA molecules;

transferring the input sample from the reverse transcription module to an amplification module within the housing, the amplification module defining a reaction volume and comprising a heater;

heating, by the heater, the input sample within at least a portion of the reaction volume to amplify the target DNA molecules within the input sample, thereby producing an output solution comprising target amplicons; and

transmitting each of the following into a detection module: A) the output solution and B) reagents formulated to produce a signal indicative of the presence of the target amplicon within the output solution, the detection module comprising a detection surface configured to retain the target amplicon to produce the signal,

the disposable molecular diagnostic test device generates the signal when the viral load of the input sample is greater than 10 copies/ml.

49. The method of claim 48, wherein the input sample is a plasma sample, the method further comprising:

separating a blood sample to produce the plasma sample comprising the target RNA molecule.

50. The method of claim 49, wherein the separating is performed within the housing of the disposable molecular diagnostic test device.

51. The method of claim 48, wherein the separating is performed outside of the housing of the disposable molecular diagnostic test device, the method further comprising:

after the separation, the plasma sample is transferred to a sample preparation module of the disposable molecular diagnostic test device.

52. The method of claim 48, further comprising:

discarding the disposable molecular test device after the readout.

53. The method of claim 48, wherein:

the signal is a visible signal; and

the detection module includes an absorbent member configured to receive the output solution generated by the amplification module and generate the visible signal.

54. An apparatus, the apparatus comprising:

a housing;

a sample preparation module within the housing, the sample preparation module defining an input reservoir configured to receive a blood sample, the sample preparation module configured to isolate a plasma sample from the blood sample, the plasma sample comprising a target RNA molecule;

a reverse transcription module within the housing configured to heat the plasma sample to produce target cDNA molecules related to the target RNA molecules, thereby producing an amplification solution; and

an amplification module disposed within the housing, the amplification module comprising a flow member defining a reaction volume configured to receive the amplification solution and a heater configured to transfer thermal energy into the reaction volume to amplify the target cDNA molecules within the amplification solution to produce an output solution comprising target amplicons.

55. The apparatus of claim 54, wherein the sample preparation module comprises a hydrophobic plasma separator.

56. The device of claim 54, wherein the sample preparation module comprises a spin-on filter configured to separate the plasma sample.

57. A method of detecting a target RNA molecule using a disposable molecular diagnostic test device, the method comprising:

transferring a biological sample to a sample preparation module within the disposable molecular diagnostic test device; and

actuating the disposable molecular diagnostic test device to cause the disposable molecular diagnostic test device to:

heating the biological sample within a reverse transcription portion of the sample preparation module to produce target DNA molecules associated with the target RNA molecules, thereby producing an amplified sample;

mixing the target cDNA with a primer composition associated with a plurality of target sequences of the target cDNA molecule;

transferring the amplified sample to an amplification module within a housing of the disposable molecular diagnostic test device;

heating the amplified sample within the amplification module to amplify the plurality of target sequences of the target cDNA molecules within the amplified sample, thereby generating an output solution comprising a plurality of target amplicons; and

transferring each of the following into a detection module within the disposable molecular diagnostic test device: A) the output solution and B) reagents formulated to produce a signal indicative of the presence of the target amplicons within the output solution, the detection module comprising a detection surface configured to retain the plurality of target amplicons within a single region to produce the signal; and

reading out the signal from the detection surface.

58. The method of claim 57, wherein the single region of the detection surface comprises a first plurality of capture probes that bind to a first target sequence of the target amplicon and a second plurality of capture probes that bind to a second target sequence of the target amplicon when the output solution is delivered into the detection module.

59. The method of claim 57, wherein the first plurality of capture probes comprises any of single-stranded nucleic acids, antibodies, or binding proteins.

60. The method of claim 57, wherein the detection surface defines at least a portion of a boundary of a detection channel through which the detection solution, the first reagent, and the second reagent are delivered.

61. The method of claim 60, wherein the detection module comprises a transparent cover opposite the detection surface, the detection channel having a depth between the cover and the detection surface of about 0.125mm to about 0.750 mm.

62. The method of claim 61, wherein the detection channel has a width of about 2mm to about 5 mm.

63. The method of claim 62, wherein the volume of the detection solution is at least 10 microliters.

64. An apparatus, the apparatus comprising:

a housing of the molecular diagnostic device; and

a reagent module disposed within the housing, the reagent module comprising a reagent housing, a reagent container containing a reagent sealed therein, a puncturer, and a deformable support member, the reagent housing defining a reagent reservoir to which the reagent is released from the reagent container when the puncturer punctures a portion of the reagent container,

the deformable support member comprises a sealing portion connected to the reagent housing to fluidly isolate the reagent reservoir, and a connecting portion connected to at least one of the piercer or the reagent container,

the deformable support member is configured to deform from a first configuration to a second configuration in response to an actuation force applied to the deformable support member, the deformable support member holding the piercer spaced apart from the portion of the reagent container when the deformable support member is in the first configuration, the piercer piercing the portion of the reagent container when the deformable support member is in the second configuration.

65. The apparatus according to claim 64, wherein said deformable support member is biased towards said first configuration.

66. The apparatus of claim 65, wherein the deformable support member exerts a biasing force on at least one of the piercer or the reagent container, the biasing force being sufficient to support the at least one of the piercer or the reagent container at a location that holds the piercer spaced apart from the portion of the reagent container.

67. The apparatus of claim 66, wherein:

the puncture device is connected in the reagent storage;

the reagent container is movably disposed within the reagent reservoir; and

the deformable support member is connected to the reagent container such that the reagent container moves from a first container position to a second container position within the reagent reservoir when the deformable support member transitions from the first configuration to the second configuration.

68. The apparatus of claim 66, wherein:

the piercer is movably disposed within the reagent reservoir; and

the deformable support member is connected to the piercer such that when the deformable support member transitions from the first configuration to the second configuration, the piercer moves from a first piercer position to a second piercer and into contact with the portion of the reagent container.

69. The apparatus of claim 64, the apparatus further comprising:

a detection module within the housing, the detection module comprising a detection surface configured to capture target molecules from a biological sample, the detection module in fluid communication with the reagent module such that a signal indicative of the presence of the target molecules is generated in response to the reagent being delivered into the detection module.

70. The apparatus of claim 69, the apparatus further comprising:

a sample preparation module within the housing, the sample preparation module defining a sample input volume to receive the biological sample and an input opening through which the sample input volume is accessible, the sample preparation module comprising a heater configured to heat the biological sample to produce an input solution; and

a cover movably coupled to the housing, the cover including a seal and a reagent actuator, the cover configured to move relative to the housing between a first cover position and a second cover position, the input opening exposed when the cover is in the first cover position, the seal of the cover covering the input opening when the cover is in the second cover position, the reagent actuator configured to cause the deformable support member to deform from the first configuration to the second configuration when the cover is moved from the first cover position to the second cover position.

71. The device of claim 69, wherein the reagent is one of a first reagent formulated to bind to the target molecule in response to the first reagent being delivered into the detection module or a second reagent formulated to generate the signal when catalyzed by the first reagent.

72. The apparatus of claim 70, wherein the second reagent is a precipitation substrate formulated to produce insoluble colored particles when the second reagent is contacted with the first reagent.

73. The apparatus of claim 69, wherein:

the reagent container is a first reagent container;

the piercer is a first piercer;

the reagent is a first reagent that is one of a catalytic reagent configured to bind to the target molecule in response to the first reagent being delivered into the detection module or a precipitating reagent configured to generate the signal when catalyzed by the catalytic reagent;

the reagent module comprises a second reagent container comprising a solution comprising a wash buffer and a blocking buffer, the blocking buffer configured to reduce the attachment of the target amplicons within the detection module away from the detection surface; and

the connecting portion of the deformable support member is connected to at least one of a second piercer or the second reagent container, the deformable support member holding the second piercer away from the second reagent container when the deformable support member is in the first configuration, the second piercer piercing the second reagent container when the deformable support member is in the second configuration.

74. A method of detecting a nucleic acid, the method comprising:

mixing a reverse transcriptase with a biological sample within a sample preparation module to form a reverse transcription solution, the reverse transcription solution being free of a ribonuclease inhibitor;

heating the reverse transcription solution to a first temperature within a lysis temperature range within a reaction volume of the sample preparation module to release ribonucleic acid (RNA) molecules;

heating the reverse transcription solution to a second temperature within a reverse transcription temperature range within the reaction volume of the sample preparation module to produce complementary deoxyribonucleic acid (cDNA) molecules, the heating to the first temperature and the heating to the second temperature occurring continuously such that the cDNA is produced in less than 1 minute of releasing the RNA molecules; and

the reverse transcription solution is transferred to an amplification module.

75. The method of claim 74, wherein said heating to a first temperature and said heating to a second temperature are performed continuously such that said cDNA is produced in less than 30 seconds of releasing said RNA molecule.

76. The method of claim 74, wherein:

the sample preparation module includes a flow member defining a flow path and a heater connected to the flow member; and

the heating to a first temperature and the heating to a second temperature are performed by activating the heater and conveying the reverse transcription solution through the flow path.

77. The method of claim 76, wherein the flow path is a serpentine flow path.

78. The method of claim 74, wherein:

the biological sample comprises MS2 phage;

the second temperature is from about 50 degrees Celsius to about 65 degrees Celsius; and

the heating to the first temperature releases the ribonucleic acid (RNA) molecule from the MS2 bacteriophage.

79. The method of claim 74, wherein:

the biological sample comprises influenza a virus;

the second temperature is from about 50 degrees Celsius to about 65 degrees Celsius; and

the heating to a first temperature releases the ribonucleic acid (RNA) molecule from the influenza A virus.

80. The method of claim 74, wherein:

the biological sample comprises hantavirus;

the second temperature is from about 50 degrees Celsius to about 65 degrees Celsius; and

the heating to a first temperature releases the ribonucleic acid (RNA) molecules from the Hantavirus.