CN108603221A - Comprehensive sample processing system - Google Patents

Abstract

A kind of comprehensive Sample purification system includes shell, sample container frame, filter mounting and cylindrical magnet.The sample container frame and filter mounting are arranged in the shell.The sample container frame is designed for keeping one or more sample containers, and filter mounting is designed for keeping one or more filters.Cylindrical magnet is close and is attached to sample container frame, and the central longitudinal axis by the motor in shell around magnet is rotated with lytic cell.It allows the interested molecule of people to be purified in lytic cell, allows the filter core of the interested molecule of people using being specifically bound to thus.The system can be easy to automate and suitable for allowing the fast purifying and analysis of the interested molecule of people such as nucleic acid and protein.

Description

Integrated Sample Processing System

technical field

The present invention generally relates to an integrated sample processing system for the isolation and/or purification of molecules of interest, such as nucleic acids and proteins, especially from intractable sample matrices and/or difficult to fragment organisms, and the use of magnetically induced vortex A method of spin-incorporating a solid monolithic filter for the easy automated separation and/or purification of nucleic acids from a sample.

Background technique

Molecular tests are the gold standard for some diagnostic tests because of their speed, sensitivity and specificity. Laboratory-developed tests (LDTs) are now one of the fastest growing segments in the in vitro diagnostics (IVD) market. Sample preparation is critical to assay reliability but often becomes a bottleneck in clinical molecular biology workflows and diagnostic assays. While there are many molecular detection modalities, there are only a handful of automated sample preparation workflow strategies. Existing instruments built around these sample preparation strategies and chemistries cost in the $17-$150k range, but they still do not provide a method for handling difficult sample matrices such as sputum and/or difficult-to-fragile organisms such as Gram Integrative approach for bacterium-positive bacteria and acid-fast bacilli (i.e., mycobacteria).

Acid-fast bacilli containing mycobacterial strains are usually isolated from the sputum of infected patients. They are called "acid-fast bacilli" because of their lipid-rich cell membrane, which is relatively impermeable to various basic dyes unless the dye is conjugated with phenol. Phlegm is thick and unmanageable. Most analytical sputum samples contain varying amounts of organic debris and various fouling normal or transient colonies. Chemical decontamination/treatment is generally used to reduce viscosity and kill contaminants while allowing recovery of mycobacteria. However, because of its unique cell membrane containing mycolic acids and high lipid content, the cells are hydrophobic and tend to clump together. This makes them impermeable to common stains such as Gram stain. Two antacid colorants are commonly used, namely carbo fuchsin and fluorescent pigments such as auramine or auramine rhodamine. Once pigmented, these cells are resistant to fading by acidifying organic solvents and are therefore termed acid-resistant. However, they remained magenta or auramine colored after sequential or simultaneous treatment with acid and alcohol.

Acid-fast smear microscopy has poor sensitivity for mycobacteria. Sensitivity of microscopy is influenced by many factors such as prevalence and severity of disease, type of sampling, quality of sampling, number of mycobacterial cells present in the sample, method of processing (direct or concentrated), centrifugation method, staining technique and test quality. It has been suggested that negative results should only be reported after examination of at least 100 (in low-income countries) and preferably 300 (in industrialized countries) microscope immersion fields (or equivalent fluorescence fields). Therefore, when performed properly, microscopy can be time-consuming and labor-intensive.

Mycobacterial strains are slow-growing bacilli with a common generation time of 12 to 18 hours. Colonies are only visible after 1 or 8 weeks of breeding time. Samples containing low concentrations of mycobacterial cells further necessitated several subcultures. Cultivation of mycobacteria on specific media allows the identification of specific mycobacterial genus contained in a biological sample. But this is time-consuming especially for those patients who are only at the beginning of the infection process.

Nucleic acid hybridization assays have been developed to detect mycobacterial strains in biological samples. Initial experiments utilized direct probe hybridization. However, the concentration of mycobacterial cells contained in samples taken from patients is usually too low to give a positive hybridization signal. Assays utilizing PCR amplification have therefore been developed. For example, commercialized as "Amplified ^TM Mycobacterium tuberculosis direct test" kit or MTD test kit The kit (Gen-Probe Inc., San Diego, CA 92121, USA) employs MtbC-specific rRNA amplification (transcription-mediated amplification) followed by amplicon detection according to the Gen-Probe HPA method (hybridization protection assay).

Given the above constraints, there is a need for a simple and efficient system that integrates sample homogenization, lysis of difficult to fragment microorganisms and polynucleotide purification to meet the needs of clinical laboratories and similar users.

Contents of the invention

In one aspect, the present application provides a comprehensive sample purification system, including a housing, a sample container rack, a filter head rack, and a cylindrical magnet. The sample container rack and filter rack are arranged in the housing. The sample container rack is designed to hold one or more sample containers and the filter head rack is designed to hold one or more filters. The cylindrical magnet is disposed proximate to and peripheral to the sample container rack and is rotatable about a central longitudinal axis of the magnet by a motor disposed within the housing.

In some embodiments, the housing includes one or more reagent racks containing one or more reagents.

In some embodiments, the system includes a plurality of said sample containers, a plurality of said filters and one or more reagent racks.

In some embodiments, the cylindrical magnet has magnetic poles arranged symmetrically about the longitudinal axis of the magnet. In other embodiments, the cylindrical magnet has opposing poles disposed on longitudinally opposite ends of the magnet. In other embodiments, the cylindrical magnet is an electromagnet.

In one embodiment, one or more sample containers are sealed and designed as a closed system after injection of one or more reagent solutions.

In one embodiment, the system further includes a reagent rack disposed within the housing, whereby the reagent rack includes reagents stored in separate sealed wells within the rack.

In use, the sample container includes a magnetic stirrer and a plurality of beads designed such that when the sample container contains cellular material and the cylindrical magnet is rotated about its longitudinal axis, the magnetic stirrer spins and agitates the beads to experience Messy mixing of cellular material allows sample homogenization and cell fragmentation.

In one embodiment, the beads comprise glass, plastic, ceramic material, mineral, metal or combinations thereof. In a particular embodiment, the beads are silica beads.

In one embodiment, the beads have a diameter in the range of 10-1000 microns.

In one embodiment, the magnetic stirrer comprises a metal or alloy. In a particular embodiment, the magnetic stirrer comprises stainless steel. In another embodiment, the magnetic stirrer comprises a polymer coated alloy core. In a particular embodiment, the magnetic stirrer comprises an alloy core coated with a polymer, whereby the alloy core comprises neodymium iron boron or samarium cobalt and/or the polymer is PTFE or parylene.

In another aspect, an automated nucleic acid purification system includes the features described above, and further includes an automated pipetting system and one or more manipulators designed to automatically dispense reagents to one or more sample containers and process the sample material and reagents in a predetermined manner. In use, the automated purification system comprises a plurality of said sample containers (each containing a stirrer and beads), a plurality of filter heads and one or more reagent racks.

In another aspect, a method for purifying a target molecule from a sample comprises the steps of: (a) providing a sample purification system according to the present disclosure; (b) placing the sample together with a magnetic stirrer and a plurality of beads in a (c) placing the sample container on a sample container rack; (d) rotating the cylindrical magnet about its longitudinal axis so that the magnetic stirrer spins and agitates the beads sufficiently to homogenize the sample and The degree to which cells in the sample are broken to form a cell lysate; (e) causing at least a portion of the cell lysate to flow through the first opening of the filter head, so that the target molecules in the cell lysate bind to the filter element in the filter head; (f) through the first opening of the filter head; an opening to expel the unbound portion of the cell lysate from the filter head, wherein the unbound portion passes through the filter element at least twice before leaving the filter head; and (g) eluting by flowing elution buffer through the first opening of the filter head Target molecules bound to the filter element and expelling elution buffer from the filter head through the first opening, wherein the elution buffer passes through the filter element at least twice before exiting the filter head.

In some embodiments, the target molecule is a polynucleotide molecule. In one embodiment, the sample comprises sputum. In certain embodiments, the sputum sample is suspected of containing Mycobacterium tuberculosis (MTB), and the method further comprises the steps of: amplifying the eluted polynucleotide molecule with primers specific for MTB, and identifying the polynucleotide molecule Does it contain MTB DNA.

In another embodiment, the target molecule purification method includes the use of an automated purification system that also includes an automated pipetting system and one or more manipulators designed to automatically dispense reagents into one or more sample containers and process them in a predetermined manner. Sample materials and reagents. In this case, each of the above steps is repeated in each of the plurality of sample containers, using an equal number of filters and one or more reagent racks.

Description of drawings

The detailed description refers to the following drawings, in which:

Figure 1 is a flow diagram illustrating one embodiment of an integrated method for lysing cells and purifying nucleic acids therefrom.

Figure 2 illustrates an exemplary single-pass nucleic acid purification system, according to one embodiment.

Figure 3 illustrates exemplary placement of magnets relative to a sample lysis chamber.

Figure 4 shows an exemplary pipette filter.

5A and 5B are schematic diagrams depicting an automated 8-channel nucleic acid purification system according to another embodiment.

Figure 6 shows a disposable transport device according to another embodiment.

Figure 7 shows an exemplary sequence of steps for MagVor (Magnetically-induced Vortexing)/filter purification of nucleic acids from sputum.

Detailed ways

In describing the preferred embodiment of the invention, specific terminology is employed for the sake of clarity. It is not intended, however, that the invention be limited to the specific terms chosen. It should be understood that each specific component includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

Figure 1 is a flowchart depicting exemplary process steps of an integrated method for lysing cells and purifying molecules of interest, such as nucleic acids or proteins therein. Method 10 includes: placing a sample tube containing a liquid sample suspension, a magnetic stirrer, and cell lysis beads on a sample holder close to the magnet (step 11); The magnet is rotated at a speed sufficient to lyse the cells in the sample suspension to homogenize the sample suspension (step 13); the homogenized sample suspension is allowed to flow through the filter matrix where the molecule of interest binds to the filter Matrix (step 15); washing the filter matrix (step 17) and eluting bound molecules of interest from the filter matrix (step 19). In some embodiments, the sample tube is preloaded with a magnetic stirrer and/or cell lysis beads and/or reagents that promote cell lysis and/or preserve the integrity of the target molecule.

The liquid sample suspension is a sample suspended in a liquid lysis medium. Exemplary samples may include biological samples, environmental samples, or non-natural samples. Exemplary biological samples may include tissue samples, biological fluid samples, cellular samples, fungal samples, protozoan samples, bacterial samples, and viral samples. A tissue sample includes tissue isolated from any animal or plant. Biological samples include but not limited to blood, cord blood, plasma, buffy coat, urine, saliva, sputum, NALC processed sputum, nasopharyngeal swab (NPS), nasopharyngeal aspirate (NPA), gastric juice, concentrated cough collection fluid, cerebrospinal fluid, oral fluid, lavage fluid (eg, bronchial), pleural fluid, stool, and leukocyte-depleted samples. Cell samples also include cultured cells, fresh or frozen cells and tissues from any cell source including fixed wax-encapsulated (FFPE) tissues. Bacterial samples include, but are not limited to, cultured bacteria, isolated bacteria, and bacteria in any of the foregoing biological samples. Virus samples include, but are not limited to, cultured viruses, isolated viruses, and viruses in any of the foregoing biological samples. Environmental samples include, but are not limited to, air samples, water samples, soil samples, rock samples, and any other samples obtained from the natural environment. Artificial samples include any sample that does not exist in the natural environment. Examples of "artificial samples" include, but are not limited to, purified or isolated material, cultured material, synthetic material, and any other man-made material.

Liquid lysis media can be isotonic, hypotonic or hypertonic. In some embodiments, the liquid lysis medium is aqueous. In certain embodiments, the liquid lysis medium contains a buffer and/or at least one salt or a combination of salts. In some embodiments, the pH of the liquid lysis medium ranges from about 5 to about 8, from about 6 to 8, or from about 6.5 to about 8.5. Various pH buffers can be used to obtain the desired pH. Suitable buffers include, but are not limited to, tris(Tris), MES, bis(2-hydroxyethylamino)tris, ADA, ACES, PIPES, MOPSO, 1,3-bis[tris] Hydroxymethylamino]propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS, N-trimethylolmethylglycine, Gly-Gly, N-bis(hydroxyethyl base) glycine and phosphate buffer (such as especially sodium phosphate or sodium potassium phosphate). The liquid lysis medium may comprise, inter alia, about 10 mm to about 100 mm buffer, about 25 mm to about 75 mm buffer, or about 40 mm to about 60 mm buffer. The type and amount of buffer used in the liquid medium can vary depending on the application. In some embodiments, the pH of the liquid lysis medium is about 7.4, which can be achieved by using about 50 mM Tris buffer. In some embodiments, the liquid lysis medium is water.

Eukaryotic cells, prokaryotic cells and/or viruses can be suspended at any suitable concentration. Preferably the sample comprises cells suspended in the liquid medium at a concentration that does not interfere with the movement of the magnetic stirrer. In some embodiments, the suspension concentration of eukaryotic cells and/or prokaryotic cells ranges especially from 1 to 1×10 ¹⁰ cells/ml, from 1 to 1×10 ⁵ cells/ml, or from 1×10 ³ to 1×10 ⁴ cells/ml. In some embodiments, the suspension concentration of virus particles ranges from 1 to 1×10 ¹³ particles/ml, 1 to 1×10 ¹⁰ particles/ml, or 1×10 ⁵ to 1×10 ⁷ particles/ml.

In certain preferred embodiments, the sample is suspected of containing MTB. In one embodiment, the sample is nasopharyngeal aspirate. In another embodiment, the sample is a nasopharyngeal swab.

As used herein, the term "cell" refers to eukaryotic cells, prokaryotic cells, viruses, endospores, or any combination thereof. Cells may thus comprise, inter alia, bacteria, bacterial spores, fungi, virions, unicellular eukaryotic organisms (such as protozoa, yeasts, etc.), isolated or aggregated cells from multicellular organisms (such as primary cells, cultured cells, tissue, whole organism, etc.) or any combination thereof.

The term "sample" refers to any material that contains or is suspected of containing a target molecule.

The term "nucleic acid" refers to individual nucleic acids and nucleic acid polymeric strands, including DNA and RNA, whether naturally occurring or synthetic (including analogs thereof) or modifications thereof, especially those known to occur in nature and Modifiers of any length.

The term "sample container" refers to an elongated, generally tubular container or vial used to secure and/or process a sample for nucleic acid purification or to contain reagents for binding to a processed sample. The sample container need not be cylindrical and may be slightly tapered along its entire length or part thereof.

The term "lyse" in relation to cells refers to the disruption of the integrity of at least a fraction of the cells to release intracellular components such as nucleic acids and proteins from the disrupted cells.

The term "homogenization" means blending or swirling (different elements such as feces, tissue, sputum, saliva) into a homogeneous mixture.

The term "closed system" or "closed container" refers to a tube or container that is sealed and operates in a substantially closed (if not completely closed) manner to impede or prevent entry (or exit) of foreign sources or foreign materials during processing ) tube or container. The components of the closure system or container may be pre-sterilized at the place of manufacture prior to use, sterilized at the point of use and/or sterilized after the respective closure system is assembled and closed prior to use.

The term "single-use disposable" means that the component part is no longer used. That is, it is disposed of after completing its intended use, ie, the processing or manufacture of the target sample.

As used herein, the term "monolithic adsorbent" or "monolithic adsorbent material" refers to a porous three-dimensional adsorbent material having a continuous interconnected pore structure within a single piece, which may comprise a rigid, self-supporting substantially monolithic The structure of the block. Monoliths are produced, for example, by casting, sintering or polymerizing precursors into a mold of the desired shape. The term "monolith adsorbent" or "monolithic adsorbent material" is to be distinguished from an aggregate of individual adsorbent particles encapsulated in a bed structure or embedded in a porous matrix, where the final product consists of individual adsorbent particles . Porous monolithic polymers are a new type of material developed in the past decade. Unlike polymers that consist of very small beads, the monolith is a single continuous polymer piece that is made using a simple molding process. The term "monolith adsorbent" or "monolithic adsorbent material" is also to be distinguished from absorbent fibers or aggregates of adsorbent-coated fibers such as filter paper or adsorbent-coated filter paper.

In one aspect, the present application provides a comprehensive sample purification system, including a housing, a sample container rack, a filter head rack, and a cylindrical magnet. The sample container rack and the filter head rack are arranged in the shell. The sample container rack is designed to hold one or more sample containers and the filter head rack is designed to hold one or more filters. The cylindrical magnet is adjacent to and external to the sample container rack and can be driven to rotate about the central longitudinal axis of the magnet by a motor disposed in the housing.

Figure 2 illustrates an exemplary single-channel sample purification system 100, according to one embodiment. System 100 in FIG. 2 includes housing 104 , sample container rack 108 , filter actuator/rack 112 and cylindrical magnet 116 . A sample container rack 108 and a filter rack 112 are disposed within the housing 104 . Sample container racks or sample container holders 108 hold sample containers 120 . The filter actuator/holder 112 in FIG. 2 is designed to hold the filter 124 attached to a syringe 176 designed such that the injection plunger 174 in the syringe 176 moves up and down to draw and dispense fluid through the filter 124 . The plunger 174 is connected to an actuator in the rack 112 that controls the movement of the plunger. A cylindrical magnet 116 is disposed proximate to and external to the sample container rack 108 and is rotated about a central longitudinal axis of the magnet 116 by a motor 130 disposed within the housing 104 .

Each sample container 120 may contain one or more lysis chambers for lysing cells in the sample. Preferably, sample container 120 (and other system components) are sealed and designed to remain a closed system before and after injection of sample 144 and/or one or more reagent solutions. Container 120 may be sealed with a lid, cap or cap. Sample container 120 can be fabricated in any suitable material, size and shape. In some embodiments, container 120 is fabricated from plastic. The interior surface of container 120 is preferably chemically inert. The sample container 120 can be, for example, in the shape of a urine collection cup, a microcentrifuge tube (such as an Eppendorf tube), a centrifuge tube, a finger tube, a microwell plate, and the like. In some embodiments, container 120 has a single cavity/compartment for holding cells 180, beads 160, and stirrer 156, as shown in FIG. In some embodiments, a container 120 may include a plurality of discrete cavities/chambers (eg, a row of wells), each capable of holding a mixture of cells 180, beads 160, and magnetic stirrer 156 in isolation from one another. In some embodiments, sample container 120 is preloaded with a magnetic stirrer and/or cell lysis beads and chemicals and/or enzymes that promote cell lysis and preserve the biological activity of target molecules.

System 100 may include a plurality of sample containers 120, a plurality of filters 124, one or more reagent racks 132, or combinations thereof. The sample container rack 108 can be designed to hold multiple sample containers 120 and can be placed on the support surface of the housing 104 to process multiple samples 144 at the same time. Likewise, the filter holder 112 can be designed to hold multiple filters 124 and can be placed on the support surface of the housing 104 to process multiple samples 144 at the same time. Sample container rack 108 may also be used as a holder for samples 144 for storage. For example, a plurality of sample containers 120 may be mounted on the sample container rack 108 and stored in a refrigerator or freezer prior to analysis.

Referring now to FIG. 3 , in use, the sample container 120 and cylindrical magnet 116 are designed such that when the cylindrical magnet 116 is rotated about its longitudinal axis, the magnetic stirrer 156 in the sample container spins sufficiently to cause the cells 180 to The forces of fragmentation and homogenization agitate the beads 160 .

The cylindrical magnet 116 may have many magnet shapes or configurations. In one embodiment, the magnet has magnetic poles (ie north and south poles) arranged symmetrically along and about the longitudinal axis of the magnet. The magnet may have a plurality of opposing poles, preferably an even number, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24, alternating around and along the longitudinal axis. In other embodiments, the cylindrical magnet has opposing poles disposed on longitudinally opposite ends of the magnet. In other embodiments, the cylindrical magnet is an electromagnet.

The magnet 116 may be rotated about an axis passing through the center of the magnet 116 above, below or beside the sample container 120 . In some embodiments, the sample container 120 is positioned perpendicular to the surface on which the sample container 120 is placed, and the magnet 116 is rotated about an axis perpendicular to the surface on which the sample container 120 is placed. In other embodiments, the sample container 120 is positioned perpendicular to the surface on which the sample container 120 is placed, and the magnet 116 is rotated about an axis parallel to the surface on which the sample container 120 is placed. In other embodiments, the sample container 120 is positioned perpendicular to the surface on which the sample container 120 is placed, and the magnet 116 is rotated about an axis at an angle to the surface on which the sample container 120 is placed. The angle is greater than 0 degrees but less than 180 degrees.

FIG. 3 shows the relative position of magnet 116 with respect to sample container 120 . Magnet 116 rotates about axis A and causes magnetic stirrer 156 within sample container 120 to rotate along axis B parallel to axis A in the same direction. Although only one axis B is shown in FIG. 3 , those skilled in the art will understand that the magnetic stirrer 156 may rotate about other B-axes parallel to the other A-axes as shown. The rotating magnetic stirrer 156 collides with the beads 160 and lyses the cells 180 in the process. The magnet 116 may be positioned alongside or above, below, or diagonally to the sample container 120 that is positioned perpendicular to the surface 190 on which the cavity or holder of the sample container 120 rests.

The sample container 120 and in particular the sample 144, the beads 160 and the magnetic stirrer 156 are located within the range of the changing magnetic field. For example, the sample container 120 may be located within the range of the rotating magnetic field, for example by placing the container 120 adjacent or close to the cylindrical magnet 116 . The changing magnetic field inter alia forces the magnetic stirrer 156 to move, such as to rotate, reciprocate, or a combination thereof, which in turn forces the beads 160, cells, and liquid medium to move. In some embodiments, sample suspension 144 is agitated with magnetic stirrer 156 at a speed and duration sufficient to lyse cells within vessel 120 . Suitable rotational speeds and durations are application dependent and can be determined empirically by one of ordinary skill in the art. In general, the rotational speed sufficient to lyse cells is determined by factors such as cell type, concentration of sample suspension 144, volume of sample suspension, size and shape of magnetic stirrer 156, amount/number of cell lysis beads 160 , size, shape and hardness, and the size and shape of the sample container 120.

In certain embodiments, the magnetic stirrer 156 is rotated at a speed between 1000-6000 rpm, preferably about 5000 rpm, for a period of 1-600 seconds, preferably about 90-120 seconds. In some embodiments, the sample container 120 (eg, in the shape of a urinalysis cup or tube) is mounted on a magnetic stirrer within the rack and stirred at a maximum set speed (>1000 rpm). In other embodiments, sample containers 120 are wells in microplates, such as ELISA plates. In other embodiments, the sample container 120 is a cylindrical container having a sample inlet and a sample outlet.

In certain embodiments, the rotational speed of magnetic stirrer 156 is increased to increase lysis efficiency and reduce the time required to achieve lysis. In certain other embodiments, the rotational speed is adjusted so that only certain types of cells are lysed. For example, in a sample suspension 144 containing multiple cell 180 types, the agitator 156 may be rotated at a first speed to lyse a first set of cells, and then rotated at a second speed to lyse a second set of cells. In other embodiments, the container 120 is coupled to a temperature control module that controls the temperature of the sample suspension 144 before, during and/or after the lysis process. In certain embodiments, the temperature of the sample suspension 144 is maintained at 2-8°C. In some embodiments, the sample suspension 144 is heated to 40-80°C, 50-70°C, or about 60°C before, during, and/or after the lysis process (eg, during rotation of the magnetic stirrer).

Magnetic stirrer 156 may be fabricated from a metal or metal alloy. In one embodiment, magnetic stirrer 156 is fabricated from stainless steel. In other embodiments, the magnetic stirrer 156 is fabricated from an alloy core coated with a chemically inert material such as a polymer, glass, or a ceramic material (eg, porcelain). Exemplary alloy core materials include neodymium iron boron samarium cobalt. Exemplary coating polymers include biocompatible polymers such as PTFE and parylene.

The magnetic stirrer 156 can be of any shape and should be small enough to be seated in the sample container 120 and move or spin or agitate within the container 120 . The magnetic stirrer 156 can in particular be a rod-shaped, cylindrical, cross-shaped, V-shaped, triangular, rectangular, rod-shaped or disk-shaped stirrer. In some embodiments, magnetic stirrer 156 has a rectangular shape. In some embodiments, the magnetic stirrer 156 has the shape of a bifurcated tuning fork. In some embodiments, magnetic stirrer 156 has a V-shape. In some embodiments, magnetic stirrer 156 has a trapezoidal shape. In certain embodiments, the longest dimension of the stirring member 156 is slightly less than the diameter of the container (eg, about 75-95% of the diameter of the container).

Cell lysis beads 160 can be any granular and/or beaded structure that is stiffer than the cells. Bead 160 may be fabricated from plastic, glass, ceramic, mineral, metal, and/or any other suitable material. In some embodiments, bead 160 may be fabricated from a non-magnetic material. Beads 160 may be rotationally symmetric about at least one axis (eg, spherical, circular, oval, elliptical, egg-shaped, and drop-shaped particles). In certain embodiments, beads 160 have a polyhedral shape. In other embodiments, beads 160 are randomly shaped particles. In some embodiments, beads 160 are raised particles. The bead 160 may especially have a diameter in the range of 10-1000 microns, 20-400 microns or 50-200 microns. The amount of beads 160 added to each lysis vessel may range, inter alia, from about 1-10000 mg, 1-1000 mg, 1-100 mg, 1-10 mg.

After the cells have been lysed, the cell lysate is drawn into a suitable filter tip 124 to allow the filter matrix 126 to which the nucleic acids are bound (see FIG. 4 ). Typically, the lysate passes through the filter base 126 at least twice before the unbound fraction exits the same end of the filter head 124. At this point, the bound nucleic acid within filter tip 124 can be stored in a sealed container for further analysis at another time. Alternatively, bound nucleic acids can be eluted from the filter using a suitable elution buffer as also described below.

Figure 4 shows an exemplary filter head. Filter tip 124 includes a porous monolithic bonded filter matrix 126 embedded in a pipette tip 127 . The monolithic bonded cartridge substrate 126 includes a monolithic sorbent or monolithic adsorbent material. Porous monoliths specifically bind nucleic acids and consist of a rigid, self-supporting, substantially monolithic structure. In some embodiments, the porous monolith does not contain additional materials that create affinity for nucleic acids. In some preferred embodiments, the porous monolith is a glass-based monolith such as a glass frit. In certain embodiments, the glass frit is a sintered glass frit. The porosity of porous monolithic materials such as glass frits or sintered glass frits can vary depending on the application. In general, the porous monolith should have a porosity that allows for the desired sample flow rate for a particular application and is capable of retaining nucleic acids within the desired size range. In some embodiments, monolithic bonded cartridge substrate 126 is a glass frit that consists of two segments (126a and 126b) with different porosities.

In some embodiments, the porous monolith is a glass frit or sintered glass frit having a porosity (i.e. average pore size) in the range of 2-400 microns, 2-300 microns, 2-220 microns, 2-200 microns , 2～180 microns, 2～160 microns, 2～140 microns, 2～120 microns, 2～100 microns, 2～80 microns, 2～60 microns, 2～40 microns, 2～20 microns, 2～16 microns , 2～10 microns, 2～5.5 microns, 4～400 microns, 4～300 microns, 4～220 microns, 4～200 microns, 4～180 microns, 4～160 microns, 4～140 microns, 4～120 microns , 4～100 microns, 4～80 microns, 4～60 microns, 4～40 microns, 4～20 microns, 4～16 microns, 4～10 microns, 4～5.5 microns, 10～400 microns, 10～300 microns , 10-220 microns, 10-200 microns, 10-180 microns, 10-160 microns, 10-140 microns, 10-120 microns, 10-100 microns, 10-80 microns, 10-60 microns, 10-40 microns , 10～20 microns, 10～16 microns, 16～400 microns, 16～300 microns, 16～220 microns, 16～200 microns, 16～180 microns, 16～160 microns, 16～140 microns, 16～120 microns , 16～100 microns, 16～80 microns, 16～60 microns, 16～40 microns, 40～400 microns, 40～300 microns, 40～220 microns, 40～200 microns, 40～180 microns, 40～160 microns , 40-140 microns, 40-120 microns, 40-100 microns, 40-80 microns, 40-60 microns, 100-400 microns, 100-300 microns, 100-220 microns, 100-200 microns, 100-180 microns , 100～160 microns, 100～140 microns, 100～120 microns, 160～400 microns, 160～300 microns, 160～220 microns, 160～200 microns, 160～180 microns, 200～400 microns, 200～300 microns Or in the range of 200-220 microns. In other embodiments, the porous monolith is a glass frit or sintered glass frit having two segments (126a and 126b) of differing porosity. Each segment may have a porosity within the above range (for example, a segment of 4-10 microns and a segment of 16-40 microns, or a segment of 16-40 microns and a segment of 100-160 microns).

In some embodiments, the thickness of the filter element is 1-30 mm, 1-25 mm, 1-20 mm, 1-15 mm, 1-10 mm, 1-8 mm, 1-6 mm, 1-4 mm, 2~30mm, 2~25mm, 2~20mm, 2~15mm, 2~10mm, 2~8mm, 2~6mm, 2~4mm, 4~30mm, 4~25mm, 4~20mm, 4~15mm, 4~10mm, 4~8mm, 4~6mm, 6~30mm, 6~25mm, 6~20mm, 6~15mm, 6~10mm, 6~8mm, 8~30mm, 8~25mm, 8~20mm, 8~15mm, 8~10mm, 10~30mm, 10~25mm, 10~20mm, 10~15mm, 15-30 mm, 15-25 mm, 15-20 mm, 20-30 mm, 20-25 mm or 25-30 mm.

In some embodiments, porous monoliths can be modified with one or more materials that have an affinity for molecules of interest, such as polynucleotides, proteins, lipids, or polysaccharides. In some embodiments, the porous monolith can be modified with one or more materials that have an affinity for nucleic acids.

In some embodiments, the filter element is fabricated from a porous glass monolith, a porous glass ceramic, or a porous polymer monolith. In some embodiments, porous glass monoliths are fabricated using sol-gel methods as described in US Pat. Nos. 4,810,674 and 4,765,818, which are hereby incorporated by reference. Porous glass-ceramics can be fabricated by means of controlled crystallization of porous glass monoliths. In preferred embodiments, the porous glass monolith, porous glass ceramic or porous monolith is not coated or embedded with any additional material such as polynucleotides or antibodies to improve its binding affinity to nucleic acids.

In some preferred embodiments, the filter element is fabricated from a fine porous glass frit that allows liquid samples to pass through. The porous glass frit is not coated or embedded with any additional material such as polynucleotides or antibodies to improve its affinity for nucleic acids or other molecules of interest. Substrates suitable for purification of nucleic acids include porous glass frits fabricated from sintered glass formed by crushing beads in a heated press to form a single monolithic structure. The uniform structure of the frit provides predictable liquid flow within the frit and allows the eluent to have similar hydrodynamics to the sample flow. Predictable liquid flow allows high recovery during elution.

Although the cartridge matrix 126 is typically housed in the pipette head 127, it can also be housed in columns, syringes or other housings of various volumes and shapes. The solution may be moved through the filter matrix 126 using a variety of devices, including manual or automated pipettes, syringes, syringe pumps, hand-held syringes, or other types of automated or manual methods for moving liquids through the filter matrix 126 .

As shown in Figures 5A and 5B, the system may further include one or more reagent racks 132 disposed within the housing. Reagent rack 132 is designed to hold one or more reagents. The reagent rack 132 may be in the form of a tray into which the reagents are poured when ready for use. In this case, the reagent may be poured into a tray to facilitate the reagent being drawn into multiple pipetting heads for delivery to multiple sample wells during sample 144 processing. Alternatively, the reagent rack 132 may be in the form of a block or a multi-well plate (eg, 24-well, 96-well, etc.) that includes a plurality of wells 152 whereby each of these wells is designed to hold any reagents for processing an individual sample 144 . In certain embodiments, wells 152 may be pre-filled with reagents and sealed within rack 132 . In some embodiments, the rack 132 is located adjacent to the sample container rack 108 and/or the filter rack 112 (FIG. 5B).

The sample purification system 100 can be operated manually, or it can be designed to operate semi-automatically or fully automatically through programmable logic. In some embodiments, the system may further include an automated pipetting system 136 (FIG. 5A) and one or more manipulators (not shown) designed to automatically transfer reagents from one or more manipulators in a predetermined computer-controlled manner. Reagent racks 132 are dispensed into a plurality of sample containers 120 and the sample material and used reagents are disposed of into appropriate waste containers 172 (FIG. 5B).

In one mode of operation, reagent rack 132 is in the form of a multi-well plate (eg, 24-well, 96-well, etc.). Preferably, the mixture is mixed using automated liquid handling as this will reduce the amount of work that needs to be done in order to prepare the mixture under investigation. Robots can also use equipment and methods known in the art to perform automated sampling experimental procedures.

Any suitable machine or device may be used to pass sample 144 through automated purification system 100 and its various processing steps. For example, the system 100 used herein can utilize a variety of robots known in the art to automate the movement of the sample 144, reagents, and other system components. Exemplary robotic systems are capable of moving a sample in one, two, or three axes and/or rotating a sample about one, two, or three axes. Exemplary robots move on rails, which may be positioned above, below, or beside the workpiece. Typically, robotic components include functional components such as manipulators that are capable of grasping and/or moving workpieces, inserting pipettes, dispensing reagents, aspirating, and the like. As used herein, "manipulator" refers to a device, preferably controlled by a microprocessor, that physically transports samples 144, containers 120, filters 124, sample container racks 108, filter racks 112, and reagent racks 132 from one location to another. a location. Each site can be a unit in the automated purification system 100 . Software for manipulator control is usually available from the manipulator manufacturer.

The robot may be translated on tracks, eg, above, below or to the side of the work area, and/or may include articulating sections that allow the manipulator to reach different locations in the work area. The robot may be driven by motors known in the art, which may be, for example, electric, pneumatic or hydraulically driven. Any suitable drive control system may be used to control the robot, such as standard PLC programming or other methods known in the art. Optionally, the robot includes a position feedback system that measures position and/or force optically or mechanically and allows the robot to be guided to a desired location. Optionally, the robot also includes position assurance mechanisms such as mechanical stops, cursors or laser guides, which allow repeated access to specific positions.

Exemplary automated sampling protocols can utilize, for example, Eppendorf epMotion5070, epMotion5075, Hamilton STARlet, STAR and STAR ⁺ liquid handling robots. Such protocols can be adapted for RNA isolation from whole blood, tissue, saliva, swabs, genomic DNA isolation, and circulating cell-free DNA such as circulating tumor DNA and circulating fetal DNA extracts and enrichments from maternal plasma .

Nucleic acid purification method

In another aspect, a method for purifying nucleic acid from a sample, comprising the steps of: (a) providing a nucleic acid purification system according to this document; (b) injecting a sample, a magnetic stirrer, and a plurality of beads into a sample container; ( c) Rotating the cylindrical magnet about its longitudinal axis so that the magnetic stirrer spins and agitates the beads to undergo a messy mixing of the cell contents to a degree sufficient to homogenize the sample and disrupt the cells in the sample to form a cell lysate (d) allowing at least a portion of the cell lysate to flow through the first opening of the filter head so that nucleic acid in the cell lysate is bound to the filter element in the filter head; (e) discharging the unbound portion of the cell lysate through the first opening into the filter head; a filter head, wherein the unbound fraction passes through the filter element at least twice before leaving the filter head; (f) by allowing the elution buffer to flow through the first opening of the filter head and draining the elution buffer from the filter head through the first opening Nucleic acids bound to the filter are eluted, wherein the elution buffer passes through the filter at least twice before exiting the filter head.

Any means for performing the method of the present application can be adopted, including fully manual, semi-automatic or fully automatic experimental operation procedures. However, the features, adaptability, simplicity, and workflow of the filter head allow for easy adjustment, automation, and efficient use with multiple clinical sample matrices, input sample volumes, and liquid handling systems. Thus, in a preferred embodiment, this mode of operation includes some automation. In one embodiment, the nucleic acid purification method includes an automated pipetting system and one or more manipulators designed to automatically dispense reagents into one or more sample containers and to dispose of sample material and reagents in a predetermined manner to Suitable single-use containers. In this case, each of the above steps is repeated with an equal number of frits and one or more reagent racks in each of the plurality of sample containers.

Samples suspected to contain MTB are potentially dangerous to users. Thus, the sample can be pre-treated by heating and/or adding reagents suitable for inactivating microorganisms present in the sample to mitigate this risk. Inactivation of microorganisms such as MTB can be performed by heating (eg 90° C. for 5 minutes) to denature active proteins, enzymatic digestion of cell wall structures, mechanical disruption to physically disrupt or inactivate cells, chemical treatment, or combinations thereof.

Chemical inactivation offers the potential to reduce or eliminate the need for heat. When processing samples for culture, simple reagents are used to digest the sputum and sterilize the samples. For cultivation, it is important to inactivate other flora present in the sputum sample so that MTB can grow without being overwhelmed by other, more rapidly growing bacteria. Preferably a decontamination or inactivation step with reagents such as sodium hydroxide (e.g. 3-5%) or cetylpyridinium chloride can be used to inactivate all other bacteria but maintain MTB with thicker and stronger cell walls Cells are intact and alive. However, depending on the method used, 20-90% of MTB cells can be killed in the process. But with respect to nucleic acid purification, the MTB cells need not be alive or intact, as long as the bacterial genomic DNA can still be amplified.

The inactivating reagent preferably allows limiting dilution of the sample and/or low pH for compatibility with silica binding. These reagents can be added to the sample at the time of collection. In some embodiments, hydrogen peroxide, alcohols such as ethanol, and o-phenylphenol (eg, 0.2-0.5%) can be used as the main active ingredient. Hydrogen peroxide (H ₂ O ₂ ) can be used as a chemical disinfectant at a concentration of 6-25% and is very stable in solution. _H2O2 is active at low pH when mixed _with 0.85% phosphoric acid. Ethanol alone (eg 95%) can inactivate MTB in sputum or water in 15 seconds. As an agricultural fungicide, o-phenylphenol is used in PHENO-CEN, SRAYPAK and CLIPPERCIDE spray fungicides at 0.1-0.41% with ethanol or isopropanol. Additionally, o-phenylphenol can be used at a low reagent-to-sample ratio within 15 minutes at room temperature and it can be used in ethanol or isopropanol or with 6.65% 2-benzyl-4-chlorophenol in Use in combination with low pH Phenolic 256 (50% to 100%).

The volume ratio of inactivating reagent to sample volume will generally be in the range of about 0.1:1 to 3:1.

Microbial inactivation within primary sample containers is important to provide a BSL-1 compliant workflow (ie, workflow that does not require biosafety cabinets). Many experimental procedures involve sample transfer prior to disinfection, which can generate aerosols and infect users. Therefore, BSL-1 compatibility requires extreme care with sample transfers prior to disinfection, especially those that can generate aerosols and infect users.

Depending on the nature of the sample, the sample may initially be liquefied to reduce its viscosity and heterogeneity for consistent sample processing. Sputum samples present a particular challenge. MTB in sputum is one of the most challenging cell and sample types to handle because of the acid-fast bacilli's lipid-rich hydrophobic cell wall and the viscous heterogeneity of sputum. Standard extraction methods for sputum generally begin with a settling process, which usually involves treatment with N-acetyl-L-cysteine (NALC) and sodium hydroxide (NaOH), followed by centrifugation, clarification, and resuspension . Thus, when dealing with highly viscous samples such as sputum, the sample can be chemically treated to reduce the viscosity so that subsequent processing steps (eg MagVor) are not affected. Exemplary mucolytic reagents for addition to samples include, but are not limited to, NALC, benzyldimethylalkylamine-trisodium phosphate (Z-TSP), benzalkonium, and Primestore (Longhorn Vaccines & Diagnostics, San Antonio Turks), It contains specific formulations for bacterial lysis and RNA and DNA stabilization. In one embodiment, sample liquefaction by chemical treatment with mucolytic agents is performed at 60°C for 20 minutes. Sputum samples from patients can generally be collected in the range of 1 to 10 milliliters, 5 to 10 milliliters or larger volumes.

At a shear rate of 90 s ^-1 , the viscosity of sputum ranges from about 100 to 6000 cP (mPa.s). Viscosity, measured in mPa·s, is determined by dividing the shear strength by the shear rate. The sample is preferably liquefied so as to reduce the viscosity of sputum by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

At least one magnetic stirrer and a plurality of cell lysis beads are present in the sample container when processing the sample. The user can simply add the sample suspension to the sample container, place the sample container close to the cylindrical magnet, and place the sample container in the sample container sufficiently by the rotating magnetic field to cause the magnetic stirrer to rotate and sufficient to homogenize and lyse the cells. Agitate the sample suspension at the speed at which the cell lysis beads are agitated.

Sample suspension, cell lysis beads, and magnetic stirrer can be placed into the sample container in any order. In some embodiments, the sample suspension is added to the sample container prior to the cell lysis beads and magnetic stirrer. In other embodiments, cell lysis beads and/or a magnetic stirrer are placed in the sample container prior to sample collection.

In certain embodiments, lysis of particular types of cells can be facilitated by adding additives to the sample suspension before and/or during the agitation step. Examples of additives include enzymes, detergents, surfactants and other chemicals such as bases and acids. Alkaline conditions (such as 10 mM NaOH) have been found to increase lysis efficiency for certain types of cells during agitation. The sample suspension may also or alternatively be heated during agitation to increase lysis efficiency. However, additives can be detrimental to downstream processing steps involving nucleic acid amplification and detection and should be eliminated where possible to simplify the process.

The stirrer/bead combination offers many advantages over traditional lysis methods. Stirrer/beads are much faster than chemical and enzymatic means and provide improved cell or virus lysis over many other types of physical lysis methods. The stirrer/bead method is also easily automated using robotics and/or microfluidics. The cylindrical magnet is reusable, does not require precise alignment of the container and can drive multiple chambers. The low cost of the magnetic stirrer allows for its single, single-use use.

Following the MagVor step, an appropriate binding buffer containing one or more solubilizers is added to the sample container to facilitate nucleic acid binding to the filter matrix 126 . The BOOM chemical or solubilizing binding of nucleic acids to silica is most efficient when the solution pH is less than 7. In this case, high ionic strength solutions containing lithium or sodium chloride or guanidinium ions are typically combined with fatty alcohols such as ethanol or isopropanol to salt out DNA and facilitate nucleic acid binding, respectively. A suitable binding buffer is employed at a concentration such that when it is added to the processed sample, the resulting volume is within the capacity of the filter head. This reduces the number of pick-and-drop cycles and thus the overall processing time.

In certain embodiments, binding buffer is added to the sample and incubated at 60°C for 10 minutes after the MagVor. In other embodiments, the solubilizer and fatty alcohol are added in the liquefaction step before the MagVor step. In other embodiments, the steps of inactivation, homogenization, and lysis are performed in a single step in as little as 15 minutes.

Exemplary solubilizers include, but are not limited to, solubilizing salts such as guanidine thiocyanate, guanidine isothiocyanate, guanidine hydrochloride, guanidine urea chloride, thiourea, sodium dodecyl sulfate (SDS), cetyl chloride Pyridine, sodium chloride, lithium chloride, potassium chloride, sodium perchlorate, lithium perchlorate, sodium iodide, and potassium iodide; fatty alcohols such as butanol, ethanol, propanol, and isopropanol; phenol and other phenolic compounds.

In some embodiments, to facilitate the selective binding of high molecular weight (HMW) nucleic acids to the filter matrix, fatty alcohols such as Isopropanol, and solubilizing salts such as guanidine isothiocyanate and/or guanidine hydrochloride are provided in the range of 1.0M to 4.0M, preferably in the range of about 3.0M to about 4.0M.

In some embodiments, fat is provided in the range of about 10% to about 25%, preferably about 15% to about 20% (optimum = 17.7%) in order to facilitate the binding (concentration) of low molecular weight (LMW) nucleic acids to the filter matrix. Alcohols such as isopropanol.

In other embodiments, to facilitate the isolation of MTB DNA from sputum, a fatty alcohol such as ethanol may be provided in the range of about 20% to about 60%, preferably about 30% to about 50% (optimum = 44%).

After the above inactivation and homogenization steps, the pH of the solution should be adjusted as necessary to obtain a pH below 7. In cases where the pH is above 7, the solution can be neutralized with a weak acid such as potassium acetate or sodium phosphate. This will be necessary when using NaOH or o-phenylphenol at pH > 11. Low pH phenolic reagents and hydrogen peroxide reagents are inherently acidic and will be least likely to require additional buffer.

In one embodiment, nucleic acid binding to the filter matrix can be accomplished by attaching the filter tip 124 to the syringe 176 with a Luer lock connection therebetween. In another embodiment, the cartridge base is in the syringe 176 . FIG. 4 shows an exemplary filter head 124 . Filter 124 includes a porous silica matrix 126 embedded within a filter body 127, an aerosol filter 128 to prevent contamination and exposure to the user, and a filter cap 129 to help maintain a closed system. In one embodiment, the cap 129 is attached to the filter head 124 with the wick. In some embodiments, the cap is a common Falcon tube cap 208 . The filter head 124 is designed to allow a liquid sample to flow through the substrate with each aspiration and dispense of the filter head 124 . Using a syringe 176 or other suitable instrument, the cell lysate in the container 120 is passed up the distal end of the filter tip 124 such that nucleic acids in the cell lysate bind to the filter matrix 126 in the pipetting tip 127 . Typically, the cell lysate is pulled up and down through the filter matrix 126 such that the lysate and unbound fraction pass through the filter matrix 126 at least twice before the unbound lysate fraction is expelled through the distal end of the pipetting tip 127 into a suitable disposable container 172. .

The combination of the sample and the solubilizing agent dehydrates the DNA and silica to facilitate adsorption onto the porous silica matrix 126 . Subsequent aspiration cycles (2-3 times) of wash buffer remove impurities from the substrate 126 . At this point, the filter tip 124 containing the nucleic acid bound to the filter matrix 126 can be stored in the sealed container 120 for further analysis at another time. Alternatively, bound nucleic acids can be eluted from the filter using a suitable elution buffer as described further below.

Nucleic acids have been shown to be extremely stable on solid supports, including silica, especially when stored in a dehydrated state without additional stabilizing agents. Thus, in another aspect, the present application provides a method of stabilizing purified nucleic acids for transport in the form of a single-use, disposable transport device 200 comprising a (eg, 50 Luer lock fitting 204 on the top side of the cap 208 of the tube) so that the filter head 124 is attached to the bottom side of the tube cap 208 ( FIG. 6 ).

Prior to use (ie, nucleic acid elution for analysis), filter tip 124 attached to cap 208 is removed from holding tube 212 and attached to syringe 176 as shown in FIG. 2 . In this case, the user can easily insert and remove the filter 124 through the luer lock connector 204 while firmly grasping the cap 208 so that the retaining tube 212 protects the user and the filter 124 from contamination. When this sequence of operations is complete, the porous silica cartridge matrix 126 with bound nucleic acid can be dried and the capped filter head 124 screwed onto the empty holding tube 212 for shipment. Retention tube 212 protects filter head 124 from contamination during transport. Stabilized nucleic acids can then be rehydrated with elution buffer and eluted into storage tubes for long-term frozen storage, or directly to detection assays using automated systems similar to those used in clinics or simply using disposable syringes 176 device or sample tube. In the latter case, the syringe 176 can be used as the mechanism for the pump-and-drop cycle of the elution buffer through the silica matrix 126 .

In preparation for molecular analysis, a pipetting cycle (2-3 times) of the elution buffer removes bound nucleic acids from the matrix 126 . Completion of this process results in nucleic acid purification in a PCR compatible buffer. This approach allows for flexibility in terms of size so that it can be used with a liquid transfer system for high flow rate applications or with simple pipetting tips for low flow rate applications.

Figure 7 shows an exemplary sequence of steps for MagVor/filter purification of nucleic acids from sputum. Sputum samples suspected of containing MTB were collected in sample containers. A chemical reagent mixture containing an inactivating reagent and a mucolytic reagent is added (step 1). The sample container is then placed on an extraction stand (or rack) and the sample contents are subjected to a magnetic vortex (MagVor) for 2-15 minutes, preferably about 10 minutes (step 2). After cell lysis, the beads were allowed to settle for 1-2 minutes and binding buffer was added to the vessel (step 3). The user attaches the filter tip to the bottom side of the cap/Luer lock in Figure 6 and connects the syringe to the top side of the cap/Luer lock using the Luer lock connection (step 4). The user penetrates the filter head through the cap from above and into the container, draws the cell lysate into the filter head and moves the syringe rod up and down into the filter matrix 2-3 times to promote binding to the filter matrix, thereby making the unbound Portion back into tube (step 4). After this binding step, the sample container is replaced with a new tube containing the wash reagent, and the filter matrix is washed with the wash buffer collected in the tube (step 5). After the washing step, the tube is lifted out of the liquid. In some embodiments, the filter head is further dried by passing air through the filter element matrix (step 6). In some embodiments, several rounds of air drying are performed to reduce residual cleaning reagents. Next, the user disengages the filter/cap adapter from the syringe, places the filter back into a new holding tube with desiccant, and couples the filter/cap adapter to the holding tube for storage (step 7) . Nucleic acids in the filter tips are stable for transport or can be eluted from the filter to purify nucleic acids for PCR analysis, etc. Elution of nucleic acids can be accomplished by passing the elution buffer through the filter matrix 2 to 3 times prior to collection.

Additives such as trehalose, 0.1% Triton-X-100 or Plus Reagent (Biomatrica) can be added to the elution buffer or to the eluted nucleic acids to enhance their stability.

In certain embodiments, the method further comprises the steps of eluting the nucleic acid, amplifying the eluted nucleic acid with primers specific for the predetermined target, and determining whether the sample contains nucleic acid corresponding to the target. Preferred targets for detection include bacterial and viral pathogens found in sputum, including but not limited to MTB, S. aureus, methicillin-resistant S. aureus (MRSA), S. pyogenes, S. pneumoniae , Streptococcus agalactiae, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Klebsiella, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter, Bordetella pertussis, meningitis Bacillus anthracis, Nocardia, Actinomyces, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella, Pneumocystis jirovecii, Influenza A virus, cytomegalovirus, and rhinovirus.

The system 100 described herein can detect MTB at levels below 1000 cells/ml, preferably below 100 cells/ml, more preferably below 50 cells/ml, most preferably below 10 cells/ml. Assuming that 1 colony forming unit (cfu) is approximately equal to 10 cells, the system described above can be used to provide detection of at least 100 cfu/ml, 10 cfu/ml, 5 cfu/ml or even 1 cfu/ml.

It should be recognized, however, that each clinical sample is unique and will differ from one another in terms of viscosity, particles, mucus, surface contaminants, microbes, and/or human genetic background. Given expected variations in clinical sample composition and the intended use of the automated filter sample preparation protocol, certain steps in the filter program may need to be modified to achieve the desired results.

For example, although the filter tips described here have larger pore sizes, sample homogenization and liquefaction are extremely important for efficient cell lysis and subsequent binding to the filter matrix. With a homogeneous and well-liquefied lysate, the sample can also flow through the filter at a higher flow rate, which reduces the total sample processing time. As shown below with the bulk plasma assay protocol, large input sample volumes can be efficiently handled with filters that provide the user with the opportunity to thoroughly homogenize and liquefy difficult samples (on-line or off-line), Only minimal attention should be paid to the input sample size.

Furthermore, it should be recognized that slower flow rates during nucleic acid binding or elution generally result in higher nucleic acid yields, albeit at the expense of overall processing time. Slower flow rates also minimize DNA shearing.

Complete drying of the filter matrix is recommended to prevent residual solvents from co-eluting with purified nucleic acid samples and inhibiting downstream processing or testing. Because the filter heads are not dried by centrifugation or vacuum filtration, it is important to maximize both the flow rate and the number of cycles during the drying step.

Because the shape, frit material, and method of attachment to the mechanical channel hand are unique to each instrument manufacturer, each liquid handling system requires a different frit configuration. Filter matrix dimensions (diameter, thickness, and pore size) correlate with nucleic acid binding capacity (and elution efficiency), as would be expected for any solid-phase extraction technique. Although thick (>4 mm) matrices can be embedded in 1 mL frits to enhance nucleic acid binding for larger samples and/or to balance matrix binding between specific frit sizes, there is a trade-off between frit thickness and the initial binding step ( There is still a trade-off between flow rates in the presence of the original lysate). Therefore, it is sometimes advantageous to embed large-diameter matrices in high-throughput frits for initial steps in automated laboratory protocols (e.g., 5 mL Hamilton/Akonni ). Assuming that there is a specific filter configuration specified by the liquid handling robot manufacturer, it is not reasonable to expect that filter nucleic acid yield will be the same across liquid handling platforms from different manufacturers or across different filter sizes. A clinical evaluation of the automated filter assay protocol and a direct comparison with commercially available automated systems is reported in detail elsewhere.

The MagVor/Filter process has many advantages over traditional methods. First, this process is compatible with automation. Second, constraining the filter matrix within the tip reduces its susceptibility to cross-contamination. On the other hand, a fibrous silica matrix on a spinning disk can easily break up and release fine particles that may be a source of pollution. Similarly, techniques that rely on the mobility of magnetic beads for purification pose similar risks. The relatively large porosity of the filter matrix allows highly viscous samples to flow through the matrix without clogging. Multiple aspiration and dispense cycles allow for enhanced target nucleic acid binding compared to centrifugation methods using a single pass of the sample through the matrix. In addition, solubilization chemistry provides robust methods for the removal of inhibitors and nucleases with long-term stability.

The invention will be further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and Tables, are hereby incorporated by reference.

example

Example 1: MagVor Homogenization and Lysis

The efficacy of the MagVor system was tested against Bacillus thuringiensis spores, Streptococcus pyogenes and MTB in sputum using a volume ratio v:v of 1:1 glass beads to sample, a total sample volume of 1 ml and 30- 120 seconds of MagVor cleavage. Extraction of MTB DNA from raw sputum is extremely challenging due to its mycobacterial cell wall and low (10 bacilli) infectious dose. Lysis potency was estimated by quantitative real-time PCR of equal sputum samples before and after MagVor treatment. MagVor treatment was found to improve nucleic acid detection by an average of 2.5 cycles (approximately 1 log) relative to untreated samples (ie, aliquots of the same sputum sample but not processed by the MagVor system).

To investigate the extent to which physical lysis kit components (particles, stirred disks, coatings) might interfere with nucleic acid extraction and purification, analyzes of four beads and three magnetic disks were performed to identify those that were not produced in solution following MagVor lysis. Lysis beads and magnetic disks for ultrafine particles. NPA samples were pooled, confirmed negative for the target DNA of interest by real-time PCR, and subsequently traced with intact methicillin MRSA or MTB target cells. Samples (0.5 ml) were processed in duplicate and lysed by 10 min MagVor treatment at 5000 rpm. Nucleic acids were purified using a manual MagVor/Filter procedure using a guanidinium binding buffer and subsequently analyzed by quantitative real-time PCR (or RT-PCR). The glass beads readily settled to the bottom of the lysis tube and did not show significant inhibition or degradation of DNA or RNA in these experiments.

As shown in Table 1, a consistent improvement in DNA recovery was obtained with MagVor treatment compared to untreated, especially at high titers.

Table 1. Combined recovery of MRSA and MTB DNA by MagVor/Filter Self-Tracing NPA:

Example 3: Comparing the Integrated MagVor/Filter Prototype with the Qiagen Nucleic Acid Purification Kit

The nucleic acid lysis and purification efficacy of the integrated system was evaluated compared to similar Qiagen nucleic acid purification kits. Model sample types included MRSA in NPA, influenza A in NPS, human genetic DNA from whole blood, and MTB in NPA. Qiagen kits include the DNA Mini Kit (no mechanical lysis, but with 10-minute proteinase K treatment), the Viral RNA Mini kit (no mechanical lysis, but with RNA vector), and the Mini Blood kit (10-minute proteinase K incubation). Since Qiagen does not have a dedicated kit for MTB extraction, the BD GeneOhm Lysis Kit was used in combination with the Qiagen Mini DNA Extraction Kit. The BD Lysis and Qiagen kits also had limited input sample volumes, therefore, NPA and NPS samples were processed in 200 µl volumes, and whole blood was processed in 100 µl, 10 µl, and 1 µl volumes. Replicate reagent plates were prepared, sealed, and processed for each sample type and titer (n = 24 extractions per sample), and purified nucleic acids were analyzed by quantitative real-time PCR comparing their average cycle threshold (Ct) with those obtained from similar Qiagen extractions (per The average period threshold obtained in n=8 samples). Positive and negative controls were performed with each plate to detect potential cross-contamination.

The results of this assay are summarized in Table 2 and demonstrate similar performance and potency relative to the Qiagen kit. For both systems, the limit of detection for MRSA in NPA and influenza A in NPS was approximately ¹⁰ cells or virus/mL. Human DNA is readily obtained from 1 microliter of whole blood, and template controls show no evidence of nucleic acid cross-contamination. These data demonstrate the scalability and efficacy of the comprehensive sample preparation prototype relative to commercially available high-quality sample preparation kits.

Table 2. Nucleic acid recovery from the MagVor-filter system (n=24) relative to other DNA extraction kits

Example 4: Liquefaction of sputum

Because most samples submitted for mycobacterial culture are contaminated with various organisms capable of rapidly growing mycobacteria prior to analysis, breath samples are generally submitted for digestion-decontamination pretreatment. Mycobacteria are then optimally recovered from clinical samples by using a protocol that releases mycobacteria trapped in mucin and cells while reducing or eliminating contaminating bacteria.

NALC-NaOH sedimentation has become the standard for decontaminating and digesting sputum samples for nontuberculous mycobacteria (NTM). However, extraction of MTB DNA from raw sputum is extremely challenging because of the high viscosity and heterogeneity of sputum on the one hand and the difficult-to-break MTB cell wall on the other. Shipping of DNA extracts reduces logistical complexity (in terms of cold shipping) compared to raw sputum. While NALC-NaOH is indeed a digestive process, NALC rapidly loses its activity, requiring daily reconstitution of fresh reagents. Furthermore, this procedure requires centrifugation, which adds additional complexity and equipment. Additionally, NaOH exposure caused MTB cell death and degraded DNA.

Therefore, it is of interest to develop a liquefaction method that can be used as an alternative option for users interested in managing sputum. Because raw sputum is a difficult sample type to transfer to sample containers, a single-step sputum liquefaction procedure using an enzyme solution was developed. By adding 1 part of liquid enzyme to 10 parts of raw sputum and incubating at 56°C for 15-20 minutes, even highly heterogeneous and viscous sputum samples were liquefied to a viscosity similar to 5-10% glycerol. These liquefied sputum samples were easily transferred and processed with the manual MagVor and filter protocol without causing the magnetic disk in the lysis tube to stop or clog the filter.

Example 5: MTB DNA extraction from raw sputum

An automated 8-channel prototype system as shown in Figures 5A and 5B was used to demonstrate the feasibility of DNA extraction from TB-positive sputum. Unidentifiable patient samples were identified by smear microscopy as either Smear2+ or Smear 4+ using the Truant TB fluorescent stain. Quartered Smear 2+ raw sputum samples and quartered Smear 4+ raw sputum samples were processed according to the liquefaction protocol in Example 4 and then added to MagVor tubes. The integrated MagVor/Filter assay protocol is followed by automated extraction/purification. The eluate was analyzed with an IS6110qPCR analyzer, and it was found that the concentration of the extract was 3.6±0.7pg/μL for Smear 2+ and 49±8pg/μL for Smear 4+. This data supports the feasibility of automated nucleic acid separators for DNA extraction from TB-positive samples.

Table 3 shows the results of a dilution series study comparing the real-time detection of MTB by the automated system and the real-time detection of MTB by the manual MagVor/filter system. MTB cells were spiked into 500 microliters of TB negative sputum and sediment (NALC-NaOH treated sputum), where 10 cells roughly equaled 1 cfu/ml. Corresponding cell grades corresponding to acid-fast bacilli (AFB) smear-positive and AFB smear-negative were included for comparison.

Table 3. Dilution series studies demonstrating real-time detection of MTB DNA by manual and automated MagVor/filter systems

The above description is to teach those skilled in the art how to implement the present invention, and does not intend to describe in detail all those obvious modifications and changes that are obvious to those skilled in the art after reading this description. However, all such obvious modifications and variations are intended to be covered within the scope of the present invention as defined by the following claims. The claims are intended to cover the claimed components and steps in any order that is effective for the purpose stated therein, unless the context clearly dictates otherwise.

Claims (20)

1. a kind of Sample purification system, including：

Shell；

The sample container frame being arranged in the shell, wherein sample container erection is calculated as keeping one or more samples Container；

The filter mounting being arranged in the shell, wherein the filter mounting is designed as keeping the one or more to include For combine allow the interested molecule of people filtration members filter；With

Cylindrical magnet close to the sample container frame and except the sample container frame, the magnet is by being arranged in the shell Motor around the magnet central longitudinal axis rotate.

2. Sample purification system according to claim 1 further includes the sample container being arranged in the sample rack, wherein the sample This container includes sample suspensions body, magnetic stirring part and multiple beads.

3. Sample purification system according to claim 1 further includes being arranged in one or more of shell reagent rack, described Reagent rack includes multiple reagents in the container for the sealing for being stored in several separations in each frame.

4. Sample purification system according to claim 1 further includes that automation aspirates system.

Further include one or more manipulators 5. Sample purification system according to claim 4, the manipulator design be for Distribute in reagent to one or more of sample containers and handle sample material and reagent automatically according to predetermined way.

6. Sample purification system according to claim 5, wherein the system further includes multiple sample containers, multiple described Filter and one or more reagent racks.

7. Sample purification system according to claim 1, wherein the cylindrical magnet has the longitudinal axis along and about the magnet The magnetic pole being arranged symmetrically.

8. Sample purification system according to claim 1, wherein the cylindrical magnet has the opposed longitudinal direction for being arranged in the magnet Opposite magnetic pole on both ends.

9. Sample purification system according to claim 2, wherein the bead is two of diameter in 10~1000 micron ranges Aoxidize silicon beads.

10. Sample purification system according to claim 2, wherein the magnetic stirring part includes the alloy core for being coated with polymer.

11. Sample purification system according to claim 10, wherein the alloy core includes neodymium iron boron or SmCo, and wherein, The polymer is PTFE or Parylene.

12. a kind of purifying the side for allowing the interested molecule of people from sample using Sample purification system according to claim 1 Method, including：

Sample tube equipped with liquid sample suspended substance, magnetic stirring part and cell cracking bead is placed on the sample rack；

By making the cylindrical magnet in the presence of the magnetic stirring part and the cell cracking bead to be enough to crack the sample The speed of cell in suspended substance rotates come the sample suspensions body that homogenizes；

In the case where allowing the interested molecule of people to be bound to filter core matrix, make the sample suspensions body to homogenize flow through to include The filter of the filter core matrix, wherein the filter is installed on the filter mounting；

Clean the filter core matrix；With

The interested molecule of people is allowed from elution of bound on the filter core matrix.

13. method according to claim 12, wherein the sample tube is preinstalled with selected from the one or more of the following group, the group by Magnetic stirring part, cell cracking bead, promote cell cracking reagent and reservation allow the interested molecule of people integrality examination Agent forms.

14. method according to claim 12, wherein described that the interested molecule of people is allowed to be nucleic acid.

15. method according to claim 14, wherein the liquid sample suspended substance includes phlegm.

16. method according to claim 15, further comprising the steps of：This is expanded with the primer for being exclusively used in mycobacterium tuberculosis The nucleic acid of elution simultaneously determines whether the nucleic acid includes Mycobacterium tuberculosis DNA.

17. a kind of method for purifying the nucleic acid from sample, including：

Sample tube is placed on sample rack, wherein the sample tube is split equipped with liquid sample suspended substance, magnetic stirring part and cell Solve bead, wherein the sample rack is located near cylindrical magnet；

The cylindrical magnet is set to be rotated around its longitudinal axis, to which the magnetic stirring part in each sample tube spins and stirs cell Cracking bead generates the degree of cell lysate to the cell fragmentation for being enough to make in sample suspensions body；

At least part of cell lysate is set to flow through the first opening of filtering pipetting head, to come from the cell cracking The nucleic acid of object is bound to the filter core matrix in the pipetting head；

By the unbonded part for flowing through the filter core matrix in flow step of the cell lysate from described in the filter First opening discharge；

As follows elution of bound to the filter core matrix nucleic acid：Elution is added by first opening through the filter Buffer solution makes the elution buffer flow through the filter core matrix, and is had passed through from filter discharge through first opening The elution buffer of the filter core matrix.

18. method according to claim 17, wherein the sample includes phlegm.

19. method according to claim 17, further comprising the steps of：This is expanded with the primer for being exclusively used in mycobacterium tuberculosis The nucleic acid of elution simultaneously determines whether the nucleic acid includes Mycobacterium tuberculosis DNA.

20. method according to claim 17, wherein the cylindrical magnet has the symmetrical cloth of longitudinal axis along and around the magnet The magnetic pole set.