WO2018167106A1 - Method and microfluidic device for processing viruses and bacteria of a sample - Google Patents

Abstract

The invention relates to a method (500) for processing viruses and bacteria of a sample by means of a microfluidic device (100). In a first step (501), the sample is washed over a retaining element (150) of the device (100), the retaining element (150) being designed to fix viruses and bacteria from the sample. In a second step (502), a first fluid is washed (502) over the retaining element (150) in order to remove viruses fixed by the retaining element (150) and to transfer the first fluid enriched with viruses into a first chamber (110) for further processing (504) of the viruses. In a third step (503), a second fluid is washed (503) over the retaining element (150) in order to remove bacteria fixed by the retaining element (150) and to transfer the second fluid enriched with bacteria into a second chamber (120) for further processing (504) of the bacteria.

Description

 description

title

 Method and microfluidic device for processing viruses and bacteria of a sample

State of the art

Microfluidic analysis systems (so-called lab-on-chips, LoCs for short) allow an automated, reliable, compact and cost-effective processing of biochemical substances for medical diagnostics, for example for the detection of pathogens in biological samples, for example in body fluids.

In many diseases such as influenza, sexually transmitted infections or respiratory tract infections, the causative agents may be of viral and / or bacterial origin. In order to obtain a holistic molecular picture of the pathogens, it is therefore necessary to identify both viral and bacterial pathogens. From the prior art, for example, Kajiura, Lauren N. et al. "Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications." Journal of Biomolecular Techniques: OVT 26.4 (2015): 118-124. PMC. Web. Feb. 24, 2017, biochemical detection protocols for the simultaneous detection of both species are known. The associated

Flow logs include a compromise between harsh ones

Lysis conditions, especially for the digestion of gram-positive bacteria, and subtle conditions to minimize damage, especially strand breaks, to viral nucleic acids. Disclosure of the invention

Advantages of the Invention Against this background, the invention relates to a method for processing viruses and bacteria of a sample with a microfluidic device. In a first step, the sample is rinsed via a retaining element of the device, wherein the retaining element is designed to fix viruses and bacteria from the sample. In a second step, a flushing of a first fluid via the retaining element for detachment from the through

 Retaining element fixed viruses and transferring the virus-enriched first fluid into a first chamber for further processing of the viruses. In a third step, a second fluid is purged via the

Retaining element for detachment of bacteria fixed by the retaining element and transfer of the bacteria-enriched second fluid into a second chamber for further processing of the bacteria.

The third step can take place before or after the second step. The purging of the first fluid and / or of the second fluid can in principle in the same direction, thus in the same flow direction, or in a different direction as the direction of the purging of the sample on the

Retaining element done. In particular, the purging of the second fluid may be in the opposite direction to the direction of purging the sample via the retention member.

By fixing viruses or bacteria by the retaining element, in particular a recording of viruses or bacteria via immobilization and / or binding, for example a chemical or electrostatic as well as positive or non-positive immobilization

or bonding with a surface of the retaining element to be understood, for example, an adsorption on the surface. Furthermore, it can be understood as a receptacle in one or more cavities, for example in the form of pores, of the retaining element. In other words, the viruses or bacteria in the rinse over the

Retaining element retained by the retaining element, in particular against the direction of the flush. As described above, the purging of the second fluid for dissolving the bacteria fixed on the retaining element may preferably be opposite to the direction of purging the sample over the

Retaining element done. This facilitates detachment of the fixed bacteria. Preferably, in the second step, the viruses are previously removed by rinsing with the first fluid in the same direction as the direction of the rinsing of the sample. The further processing of the viruses or bacteria can be part of a detection method of the viruses or bacteria, in particular with the aid of a polymerase chain reaction, which is carried out, for example, in the microfluidic device set up for this purpose.

The method according to the invention has the advantage that viruses and bacteria can be isolated particularly efficiently and in a particularly simple manner from the sample for further, separate processing. The viruses and bacteria may in particular be pathogens of

For example, flu, sexually transmitted infections or

Respiratory tract infections act. In particular, by the invention

advantageously no compromise between harsh lysis conditions for bacteria and subtle conditions for the further processing of the viruses required because of the advantageous isolation a separate

Further processing of bacteria and viruses from the sample is possible. Thus, advantageously no loss of the yield of processable viruses or bacteria due to the compromise described must be accepted.

In a particularly advantageous embodiment of the invention further processing of the viruses comprises lysis of the viruses or denaturation of protein envelopes of the viruses. Furthermore, the processing may include performing a polymerase chain reaction (PCR) of RNA or DNA, optionally after

Use of a reverse transcriptase to transcribe viral RNA into DNA comprising viruses. This processing may preferably take place in the first chamber, wherein in the first chamber preferably for the other

Processing are required upstream of required substances, for example in freeze-dried form as a lyophilizate. This has the advantage that the more Processing of the virus can take place immediately after isolation from the sample without the viruses must first be transported. The upstream substances may in particular comprise substances for lysing the viruses or denaturing proteins of the viruses, as well as substances for carrying out a (reverse transcriptase) polymerase chain reaction of the viruses, ie in particular substances adapted to the further processing of viruses. Furthermore, the substances may include enzymes, primers and probes for detecting genetic features of the viruses.

According to a further particularly advantageous embodiment, the further processing of the bacteria comprises carrying out a polymerase chain reaction of DNA of the bacteria, preferably in the second chamber of the device, wherein required in the second chamber for further processing

Preceding substances, for example in freeze-dried form as a lyophilizate. This has the advantage that the further processing of the bacteria can take place immediately after isolation from the sample, without the bacteria first having to be transported further. The upstream substances may in particular substances for carrying out the

Polymerase chain reaction of the bacteria include, ie in particular on the further processing of bacteria tuned substances. Further, the substances may comprise enzymes for detecting genetic characteristics of the bacteria.

According to an advantageous embodiment of the invention, the sample can be mixed before the rinsing of the sample via the retaining element with a binding buffer for binding the virus to the retaining element. This advantageously increases the proportion of viruses isolated from the sample.

According to a particularly advantageous development of the invention, before rinsing with the second fluid, lysis of the bacteria bound on the retaining element takes place. This has the advantage of being a part of

Further processing of the bacteria already on the retaining element and thus the process can be performed faster and more efficient overall. In one embodiment of this development, the retention element for lysing the bacteria is exposed to a lysis buffer, in particular during a predetermined period of time. The lysis buffer can be substances for chemical lysis of the bacteria such as chaotropic salts and

Include proteinase K. Supportive or alternatively that can

Retaining element to be exposed to ultrasound for lysing the bacteria. In this case, the retaining element can be surrounded with a fluid comprising water, in particular flooded, to the liberated DNA by lysis in a neutral medium without inhibiting substances for a

to be able to record subsequent polymerase chain reaction. In a particularly advantageous development of the invention, this includes

Retaining a filter, wherein at least a portion of the surface of the filter comprises a material for fixing, in particular binding of nucleic acids and / or predetermined proteins. For example, the filter may be a silica filter or a silica membrane. This has the advantage that the complexity of the microfluidic device is low, since the specific retention of the two species, viruses and bacteria, is achieved by two different properties of the retention element, namely the retention of bacteria via size exclusion and the retention of viruses via affinity binding. Thus, with a silica filter, a complex functionality of the device can be achieved with minimal space. Especially with a

Filter as a retaining element, the flushing of the second fluid can be carried out preferably in the opposite direction to the direction of the flushing of the sample via the retaining element, in particular for detachment of the bacteria fixed on the filter. This causes an advantageously easier detachment.

In a particular embodiment of the invention, a selective lysis of human cells in the sample is carried out before the rinsing of the sample via the retaining element. This is particularly advantageous in the case of a sample in which viruses can be present in human host cells.

The invention also provides a microfluidic device for

Processing of viruses and bacteria of a sample. The device comprises a retaining element, wherein the retaining element is designed for fixing viruses and bacteria from the sample. Furthermore, the device comprises a first chamber for receiving fixed by the retaining element viruses and a second chamber for receiving bacteria fixed by the retaining element, wherein the first chamber comprises a first reaction mix for further processing of the viruses and the second chamber comprises a second reaction mix for further processing of the bacteria.

Under a reaction mix can be understood in particular a substance mixture with substances for the further processing of viruses or bacteria, such as the above-mentioned substances for a

Polymerase chain reaction or enzymes for detection of genetic traits.

For the advantages of the device and its developments below, reference is made to the corresponding above-mentioned advantages of the disclosed method according to the invention.

In a particularly advantageous embodiment of the device, the retaining element comprises a filter, wherein at least a part of the surface of the filter comprises a material for fixing, in particular binding of nucleic acids and / or predetermined proteins.

According to an advantageous embodiment, the device comprises a first valve and a second valve for the fluidic separation of the first chamber and the second chamber from the retaining element. This has the advantage that access to the two chambers only after fixing the bacteria and viruses to the retaining element allows and thus a

Contamination of the two chambers can be prevented in particular by other components of the sample.

In a further advantageous embodiment of the invention, the

Device an ultrasonic source or an interface, in particular a

Membrane, for an ultrasonic source, the ultrasonic source

or the interface are arranged next to the retaining element. This has the advantage of having an immediate impact of

Ultrasonic energy to the retaining element for effective lysis of cells, especially bacteria, is made possible. According to a further advantageous embodiment of the invention, the device comprises a heater disposed adjacent to the retaining element, for example, a resistance heater or a Peltier element. As a result, a thermal lysis of the bacteria on the retaining element can advantageously be carried out.

Brief description of the drawings

Embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description.

Show it

Figure 1 shows an embodiment of the device according to the invention and

2 shows a flowchart of an associated embodiment of the method according to the invention.

Embodiments of the invention

FIG. 1 shows an exemplary embodiment of the microfluidic device 100 according to the invention. FIG. 2 shows a flow chart 500 of an associated microfluidic device 100

Embodiment of the method 500 according to the invention.

The microfluidic device 100 shown in FIG

Retaining member 150, wherein the retaining member 150 for fixing viruses and bacteria from a sample, for example, a sample of human body fluid such as blood or sputum is formed. For example, the retaining element 150 comprises a filter 151, wherein at least part of the surface of the filter 151 comprises a material for fixing nucleic acids and / or predetermined proteins. It may be a silica filter with exemplary pore sizes between 0.025 and 10 microns, preferably between 0.1 and 1 microns. Furthermore, the device 100 comprises a first chamber 110 for receiving viruses bound by the retaining element 150 and a second chamber 120 for receiving bacteria bound by the retaining element 150. The two chambers 110, 120 may be fluidically connected to the retaining element 150 via a first channel 131, the device 100 in this example having a first valve 191 and a second valve 192 for fluidically separating the first chamber 110 and the second chamber 120 from the retaining element 150 includes. In addition, a connection 130 may be lockable from the retaining element 150 to the first channel 131 via a third valve 193.

For example, the sample can be introduced via a closable opening 160 of the device 100 into a second channel 132 connected to the retaining element 150, wherein the retaining element 150 can be fluidically separated from the second channel 132 via a fourth valve 194.

Preferably, the first chamber 110 comprises a first reaction mix 111 for further processing of the viruses and the second chamber 120 a second reaction mix 121 for further processing of the bacteria. In particular, the first and second reaction mixes 111, 121 can be viruses

or contain bacteria matched typical substances for the preparation and / or conduct of a polymerase chain reaction, such as polymerase. In particular, the first reaction mix 111 may comprise substances for a reverse transcriptase polymerase chain reaction (RT-PCR), for example a QIAGEN® OneStep RT-PCR kit or a NEB® OneTaq® RT-PCR kit, for the RNA of the virus for further

Detection reactions, preferably a polymerase chain reaction, first to rewrite in cDNA. Both the first and the second reaction mix can be in freeze-dried form, so-called lyophilisate. Furthermore, the reaction mixtures may comprise enzymes, primers or probes for detecting genetic characteristics of the bacteria or viruses.

As indicated above, FIG. 2 shows a flowchart 500 of a

Embodiment of the method according to the invention, for example Use of the embodiment of the inventive device 100 according to FIG. 1. In a first step 501, the sample is passed over the

Retention element 150, in this example the above-mentioned silica filter, the device 100 rinsed to fix the viruses and bacteria from the sample on the filter. Before a rinse of the sample, the sample can with a

Binding buffer for binding the virus to the retaining element 150 are mixed. The binding buffer may be, for example, a

Phosphate buffer having a pH around 5.5, which may contain between 1 and 3 molar guanidinium hydrochloride or guanidinium thiocyanate, preferably between 1.75 and 2.25 M guanidinium hydrochloride or

 Guanidine thiocyanate. Preferably, the binding buffer is acid-buffered (pH <= 6) to promote interaction between silanol groups of the silica filter and the sugar-phosphate residue of the DNA. Further, the binding buffer may contain ethanol for the precipitation of the DNA. The fixation of the bacteria takes place via a size exclusion, that is, passages, in particular pores, of the filter 150 are smaller than the size of the

to be withdrawn bacteria.

In particular, when human cells are present in the sample, prior to rinsing the sample via the retention element, selective lysis of these human cells may occur since viruses to be detected may be contained in the human cells. Such selective lysis can be achieved, for example, with the use of 0.05% TWEEN® 80 (final concentration) or 0.2% saponin (final concentrations). As a result of this selective lysis, intact bacteria and virus present in solution are obtained in the midst of

Human cell lysates and denatured proteins in the sample.

In a second step 502, a flushing of a first fluid via the retaining element 150 is carried out to detach viruses bound by the retaining element 150 and to transfer the virus-enriched fluid into the first chamber 110 for further processing of the viruses. The first fluid may, in particular, be an elution buffer for detaching the viruses from the retention element 150. In particular, the elution buffer may comprise water, preferably double-distilled water. Alternatively, an elution buffer can be used which is based on a mixture of Tris (hydroxymethyl) aminomethane and hydrochloric acid, present in a molarity of between 3 and 7 millimolar, ideally 5 millimolar and having a pH of 8.5.

In a third step 503, a second fluid is flushed via the retention element 150 to detach bacteria bound by the retention element 150 and transfer the bacteria-enriched fluid into the second chamber 120 for further processing of the bacteria. The second fluid may in particular also be an elution buffer, it being possible, for example, to use a similar elution buffer as in the second step described above. If, as in this embodiment, the bacteria are retained by a filter 150 from the sample and thereby fixed on the filter 150, the flushing of the second fluid through the filter, preferably against the direction of the sample's rinse, can effectively effect the bacteria from the filter 150 to solve again.

Preferably, prior to rinsing 503 with the second fluid, lysis of the bacteria bound on the retention member 150 may occur. Lysis can take place chemically, thermally and / or via the action of ultrasound.

For example, the device 100 may comprise an ultrasound source 170 or an interface 170, in particular a membrane, for example an elastic polymer membrane, for an ultrasound source, wherein the

Ultrasonic source 170 and the interface 170 for a direct action of ultrasound next to the retaining element 150 are arranged. In addition, the retaining element 150 can thereby with a water

surrounding fluid for receiving the released by the lysis DNA, for example, be flooded.

Alternatively or additionally, a chemical lysis of the bacteria on the retaining element 150 can also be carried out using a lysis buffer before rinsing 503. For example, the lysis buffer may comprise detergents such as Tween® or Triton®-X and / or ethylenediaminetetraacetic acid, EDTA for short. To the inhibitory effect of EDTA for a To neutralize subsequent polymerase chain reaction can be added after lysis magnesium chloride, preferably in one

Minimum concentration of 2.5 millimolar, preferably at least 3 millimolar magnesium chloride. This additional magnesium chloride is similar to EDTA inactivated magnesium chloride in a typical reaction mix for one

Polymerase chain reaction. This has the advantage that the lysate can be transferred into the second chamber 120 for a polymerase chain reaction without DNA purification.

Furthermore, a thermal lysis of the bacteria alone or in

Combined with a chemical lysis and / or an ultrasonic lysis. For this purpose, the device 100 may be adjacent to the retaining element 150

arranged heating element 180 include, for example, a

Resistance heating or a Peltier element.

After lysis, it is not necessary to flush the filter 150 in the opposite direction as described above, since the DNA released from the lysed bacteria can also be flushed through the filter 150 if the pores of the filter 150 are not too small.

As described above, after the third step 503, the other

Processing of the Viruses or Bacteria as a Fourth Step 504 Lysis of the viruses or denaturation of protein envelopes of the viruses followed by a polymerase chain reaction of nucleic acid sections of the viruses or of the bacteria takes place, for example for detection of the viruses or of the bacteria, in particular in the first chamber 110 and in the second chamber 120th

Claims

claims A method (500) for processing viruses and bacteria of a sample with a microfluidic device (100), comprising the steps of: Rinsing (501) the sample via a retaining element (150) of the device (100), wherein the retaining element (150) is designed to fix viruses and bacteria from the sample. Purging (502) a first fluid via the retention member (150) to  Detachment of viruses fixed by the retention element (150) and transfer of the virus-enriched fluid into a first chamber (110) for further processing (504) of the viruses. Purging (503) a second fluid via the retention member (150) to detach bacteria fixed by the retention member (150) and transferring the bacteria-enriched fluid into a second chamber (120) for further processing (504) the bacteria. The method (500) of claim 1, wherein the purging (502, 503) of the first fluid and / or the second fluid is in the opposite direction to the direction of purging (501) of the sample via the retention member (150). 3. The method (500) according to claim 1 or 2, wherein before rinsing (503) with the  second fluid is a lysis of the on the retaining element (150) fixed bacteria. 4. The method (500) according to claim 3, wherein for lysing the bacteria the Retaining element (150) is exposed to a lysis buffer. 5. The method (500) of claim 3 or 4, wherein for the lysing of the bacteria, the retaining element (150) is subjected to ultrasound, preferably after a suspension, in particular flooding, of the retaining element (150) with a fluid comprising water. The method (500) of any one of the preceding claims, wherein the sample is mixed with a binding buffer to bind the viruses to the retention member (150) prior to rinsing (501) the sample via the retention member (150). 7. The method (500) according to any one of the preceding claims, wherein the further  Processing (504) the virus comprises lysis of the viruses or denaturation of protein envelopes of the viruses. 8. The method (500) according to any one of the preceding claims, wherein the further  Processing (504) of viruses or bacteria performing a  Polymerase chain reaction of DNA of viruses or bacteria. 9. The method (500) according to any one of the preceding claims, wherein the  Retaining element (150) comprises a filter (150), wherein at least a part of the surface of the filter (150) comprises a material for fixing, in particular binding of nucleic acids and / or predetermined proteins. 10. The method (500) according to any one of the preceding claims, wherein prior to the rinsing (501) of the sample via the retaining element (150), a lysis of human cells in the sample takes place. 11. IVlikrofluidische device (100) for processing viruses and bacteria of a sample, characterized in that the device (100) comprises a retaining element (150), wherein the retaining element (150) for fixing viruses and Bacteria is formed from the sample, and that the device comprises a first chamber (110) for receiving by the retaining element (150) fixed viruses and a second chamber (120) for receiving by the retaining element (150) fixed bacteria, said first chamber (110) a first reaction mix (111) for further processing of the viruses and the second chamber (120) comprises a second reaction mix (121) for further processing of the bacteria. The microfluidic device (100) of claim 11, wherein the device (100) includes a first valve (191) and a second valve (192) for fluidly isolating the first chamber (110) and the second chamber (120) from the retention member (150) ). 13. The microfluidic device (100) according to claim 11 or 12, wherein the  Retaining element (150) comprises a filter (150), wherein at least a part of the surface of the filter (150) comprises a material for fixing, in particular binding of nucleic acids and / or predetermined proteins. 14. The microfluidic device (100) according to one of claims 11 to 13, wherein the device (100) comprises an ultrasound source (170) or an interface (170), in particular a membrane, for an ultrasound source, wherein the  Ultrasonic source (170) or the interface (170) next to the  Retaining element (150) are arranged. The microfluidic device (100) of any one of claims 11 to 14, wherein the device (100) comprises a heating element (180) disposed adjacent the retention member (150).