DE102017204195A1 - 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 with a microfluidic device (100). In a first step (501), the sample is rinsed via a retaining element (150) of the device (100), wherein the retaining element (150) is designed to fix viruses and bacteria from the sample. In a second step (502), flushing (502) of a first fluid via the retention member (150) to release viruses fixed by the retention member (150) and transfer of the virus-enriched first fluid into a first chamber (110) for one further processing (504) of the viruses. In a third step (503), flushing (503) of a second fluid via the retention member (150) to release bacteria fixed by the retention member (150) and transfer of the bacteria-enriched second fluid into a second chamber (120) for one further processing (504) of the bacteria.

Description

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: JBT 26.4 (2015): 118-124. PMC. Web. 24 Feb 2017 , biochemical detection protocols are known for the simultaneous detection of both species. The associated sequence protocols comprise a compromise between harsh lysis conditions, in particular for the digestion of gram-positive bacteria, and subtle conditions in order to minimize damage, in particular 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 of viruses fixed by the retaining element and transfer of the virus-enriched first fluid into a first chamber takes place for further processing of the viruses. In a third step, a second fluid is flushed via the retaining element to detach bacteria fixed by the retaining element and to transfer 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 flushing of the first fluid and / or of the second fluid can in principle take place in the same direction, thus in the same flow direction, or in a different direction as the direction of the flushing of the sample via the retaining element. 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.

A fixation of viruses or bacteria by the retaining element can be understood in particular to include viruses or bacteria via immobilization and / or binding, for example chemical or electrostatic and positive or non-positive immobilization or bonding to a surface of the retaining element, for example 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 are retained during the flushing via the retaining element by the retaining element, in particular counter to the direction of the flushing. As described above, the flushing of the second fluid for releasing the bacteria fixed on the retaining element can preferably take place counter to the direction of the flushing of the sample via the retaining element. 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, influenza, sexually transmitted infections or respiratory tract infections. In particular, no compromise between harsh lysis conditions for bacteria and discreet conditions for the further processing of viruses is advantageously required by the invention, since due to 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 a Denaturation of protein envelopes of viruses. Further, the processing may include performing a polymerase chain reaction (PCR) of RNA or DNA, optionally after employing a reverse transcriptase to transcribe viral RNA into DNA of the virus. This processing may preferably take place in the first chamber, wherein in the first chamber, substances required for further processing are preferably arranged upstream, for example in freeze-dried form as lyophilisate. This has the advantage that the further processing of the viruses can take place immediately after isolation from the sample, without the viruses having to be transported further first. The upstream substances may in particular comprise substances for lysis of the viruses or denaturation of 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 in the second chamber required for further processing substances are upstream, for example in freeze-dried form as lyophilisate. 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 comprise substances for carrying out the polymerase chain reaction of the bacteria, ie in particular substances adapted to the further processing of bacteria. 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 that part of the 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 may comprise substances for chemical lysis of the bacteria, such as, for example, chaotropic salts and proteinase K. Alternatively or alternatively, the retention member may 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, in order to be able to take up the DNA liberated by the lysis in a neutral medium without inhibiting substances for a subsequent polymerase chain reaction.

In a particularly advantageous embodiment of the invention, the retaining element comprises a filter, wherein at least part 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. Particularly in the case of a filter as a retaining element, the flushing of the second fluid can preferably take place in the opposite direction to the direction of the flushing of the sample via the retaining element, in particular for the 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 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 viruses fixed by the retaining element and a second chamber for receiving bacteria fixed by the retaining element, the first chamber having a first reaction mix for further processing of the viruses and the second chamber a second reaction mix for further processing the bacteria includes.

A reaction mixture may, in particular, be understood as meaning a mixture of substances with substances for the further processing of the viruses or bacteria, for example the abovementioned substances for a polymerase chain reaction or enzymes for detecting genetic features.

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

In a further advantageous development of the invention, the device comprises an ultrasound source or an interface, in particular a membrane, for an ultrasound source, the ultrasound source or the interface being arranged next to the retaining element. This has the advantage that an immediate effect of ultrasonic energy on 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.

list of figures

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

• 1 ein Ausführungsbeispiel der erfindungsgemäßen Vorrichtung und
• 2 ein Flussdiagramm eines zugehörigen Ausführungsbeispiels des erfindungsgemäßen Verfahrens.

Show it

• 1 an embodiment of the device according to the invention and
• 2 a flowchart of an associated embodiment of the method according to the invention.

Embodiments of the invention

1 shows an embodiment of the microfluidic device 100 according to the invention. 2 shows a flowchart 500 an associated embodiment of the method according to the invention 500 ,

In the 1 illustrated microfluidic device 100 includes a retaining element 150, wherein the retaining element 150 for fixing viruses and bacteria from a sample, such as a sample of human body fluid such as blood or sputum is formed. For example, the retaining element comprises 150 a filter 151 , wherein at least a part of the surface of the filter 151 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 comprises 100 a first chamber 110 for receiving by the retaining element 150 bound viruses and a second chamber 120 for receiving by the retaining element 150 bound bacteria. The two chambers 110 . 120 can have a first channel 131 be fluidly connected to the retaining element 150, wherein the device 100 in this example, a first valve 191 and a second valve 192 for the fluidic separation of the first chamber 110 or the second chamber 120 from the retaining element 150 includes. In addition, also a connection 130 from the retaining element 150 to the first channel 131 over a third valve 193 be lockable.

For example, the sample may have a closable opening 160 the device 100 in one with the retaining element 150 connected to the second channel 132, wherein the retaining element 150 from the second channel 132 via a fourth valve 194 can be separated fluidly.

Preferably, the first chamber comprises 110 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 the second reaction mix 111 . 121 contain viruses or bacteria matched typical substances for the preparation and / or conduct of a polymerase chain reaction, such as for example, polymerase. In particular, the first reaction mix 111 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, comprise the RNA of the viruses for further detection reactions, preferably a polymerase chain reaction , first into cDNA rewrite. 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 stated above, shows 2 a flowchart 500 an embodiment of the method according to the invention, for example using the embodiment of the device according to the invention 100 according to 1 , In a first step 501 The sample is rinsed via the retention member 150, in this example the above-mentioned silica filter, of the apparatus 100 to fix the viruses and bacteria from the sample to the filter. Before rinsing the sample, the sample can be used with a binding buffer to bind the virus to the retention element 150 be mixed. The binding buffer may be, for example, a phosphate buffer with 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 guanidinium 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 chosen smaller than the size of the bacteria to be backed up.

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 middle of the human cell lysate and denatured proteins in the sample.

In a second step 502 Rinsing of a first fluid via the retaining element 150 occurs to detach from the retaining element 150 bound viruses and transfer of 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 act. In particular, the elution buffer may comprise water, preferably double-distilled water. Alternatively, an elution buffer based on a mixture of tris (hydroxymethyl) aminomethane and hydrochloric acid, in a molarity of between 3 and 7 millimolar, ideally 5 millimolar, and having a pH of 8.5 may also be employed.

In a third step 503 Rinsing of a second fluid via the retaining element 150 occurs to detach from the retaining element 150 bound bacteria and transfer of 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 pass through a filter 150 be retained from the sample and thereby on the filter 150 are fixed, the flushing of the second fluid through the filter can preferably be carried out counter to the direction of the flushing of the sample to effectively remove the bacteria from the filter 150 to solve again.

Preferably, before rinsing 503 with the second fluid, a lysis of the on the retaining element 150 bound bacteria take place. Lysis can take place chemically, thermally and / or via the action of ultrasound.

For example, the device 100 an ultrasound source 170 or an interface 170, in particular a membrane, for example an elastic polymer membrane, for an ultrasound source, the ultrasound source 170 or the interface 170 for direct exposure to ultrasound adjacent to the retention element 150 are arranged. In addition, the retaining element 150 thereby be surrounded with a fluid comprising water 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 using a lysis buffer before rinsing 503 be performed. For example, the lysis buffer may comprise detergents such as Tween® or Triton®-X and / or ethylenediaminetetraacetic acid, EDTA for short. To neutralize the inhibitory effect of EDTA for a subsequent polymerase chain reaction after lysis, magnesium chloride may be added, preferably at a minimum concentration of 2.5 millimolar, preferably at least 3 millimolar magnesium chloride. This additional magnesium chloride compensates for EDTA inactivated magnesium chloride in a typical polymerase chain reaction reaction mix. This has the advantage that the lysate without DNA purification in the second chamber 120 can be converted for a polymerase chain reaction.

Furthermore, a thermal lysis of the bacteria can be carried out alone or in combination with a chemical lysis and / or an ultrasound lysis. This can be done by the device 100 one next to the retaining element 150 arranged heating element 180 include, for example, a resistance heater or a Peltier element.

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

As described above, after the third step 503 the further processing of viruses or bacteria as a fourth step 504 lysis of the viruses or denaturation of protein shells of the viruses followed by a polymerase chain reaction of nucleic acid sections of the viruses or of the bacteria take place, for example for detection of the viruses or of the bacteria, in particular in the first chamber 110 or in the second chamber 120 ,

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• Kajiura, Lauren N. et al. "Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications." Journal of Biomolecular Techniques: JBT 26.4 (2015): 118-124. PMC. Web. 24 Feb 2017 [0002]

Claims (15)

Method (500) for processing viruses and bacteria of a sample with a microfluidic device (100), comprising the steps: 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. Flushing (502) a first fluid via the retention member (150) to detach viruses fixed by the retention member (150) and transferring the virus-enriched fluid into a first chamber (110) for further processing (504) 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. Method (500) 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). Method (500) Claim 1 or 2 wherein, prior to rinsing (503) with the second fluid, lysing of the bacteria fixed on the retention member (150) occurs. Method (500) Claim 3 wherein, for lysing the bacteria, the retention member (150) is exposed to a lysis buffer. Method (500) Claim 3 or 4 in that, for lysing the bacteria, the retention element (150) is subjected to ultrasound, preferably after a suspension, in particular flooding, of the retention element (150) with a fluid comprising water. The method (500) of any 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). The method (500) of any one of the preceding claims, wherein the further processing (504) of the viruses comprises lysis of the viruses or denaturation of protein envelopes of the viruses. The method (500) of any one of the preceding claims, wherein the further processing (504) of the viruses or bacteria comprises performing a polymerase chain reaction of DNA of the viruses or bacteria, respectively. Method (500) according to 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 fixation, in particular binding of nucleic acids and / or predetermined proteins. Method (500) according to one of the preceding claims, wherein prior to the rinsing (501) of the sample via the retaining element (150) lysis of human cells in the sample takes place. A microfluidic 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) is designed to fix viruses and bacteria from the sample, and the apparatus comprises a first chamber (110) for receiving viruses fixed by the retaining element (150) and a second chamber (120) for receiving bacteria fixed by the retaining element (150), the first chamber (110) having 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. Microfluidic device (100) according to Claim 11 wherein the device (100) comprises a first valve (191) and a second valve (192) for the fluidic separation of the first chamber (110) and the second chamber (120) from the retaining element (150). 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 fixation, in particular binding of nucleic acids and / or predetermined proteins. 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, the ultrasound source (170) or the interface (170) being arranged next to the retaining element (150). Microfluidic device (100) according to one of Claims 11 to 14 wherein the device (100) comprises a heating element (180) arranged adjacent to the retaining element (150).

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