Method for and carrying out parallel nucleic acid amplifications

Abstract

The invention relates to a method for carrying out parallel nucleic acid amplifications, in particular parallel polymerase chain reactions, using a microfluidic device (100).

Description

[0001] Description

[0002] title

[0003] METHOD AND PROCEDURE OF PARALLEL NUCLEAN ACID AMPLIFICATIONS

[0004] State of the art

[0005] The detection of pathogens in a biological sample can be achieved by identifying fragments of the pathogens' nucleic acids within the sample. This is done using nucleic acid amplification, a process in which nucleic acids are amplified section by section, to specifically search for these fragments. Such methods can be used at the point of care and as immediate, near-patient diagnostics, particularly in doctors' offices and hospitals, with microfluidic systems. In this process, a patient sample is introduced into a microfluidic cartridge, which is then activated in an analyzer to perform nucleic acid amplification within the cartridge.

[0006] In the presence of certain symptoms, it is preferable to test a patient sample at the point of care for several potential pathogens in parallel, without having to significantly extend the required treatment time.

[0007] Disclosure of the invention

[0008] Advantages of the invention

[0009] Against this background, the invention relates to a method for performing parallel nucleic acid amplifications, in particular for the detection of pathogens, using a microfluidic device. The device according to the invention can, in particular, be designed as a microfluidic cartridge which can be processed with an analyzer, for example, based on a cartridge and an analyzer as described in documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1. The invention thus also relates to a microfluidic system with such a cartridge and such an analyzer.

[0010] In the presented method, a sample is first taken into the microfluidic device, for example by introducing the sample into an input chamber of the device.

[0011] The sample is, in particular, a biological sample containing nucleic acids, i.e., a sample from a living organism, especially comprising a body fluid or a part of a body tissue, for example, blood, sputum, cerebrospinal fluid, urine, or a swab. The method is preferably used to detect a microorganism, especially a pathogen such as bacteria, viruses, or fungi, in the sample. After collection, the sample can be transferred to a medium for transport from the living organism to the analysis, i.e., to a microfluidic device. The transport medium preferably comprises a medium without lysing properties to keep the cells intact, which is also referred to as a stabilizing transport medium and which also allows the intact cells to be transferred with the transport medium to a nutrient medium for cell cultivation.According to a particular embodiment, the transport medium can contain a nutrient medium or culture medium. At least some of the cells are separated from the sample using a solid phase, preferably via size exclusion. The solid phase can preferably be a filter. Alternatively or additionally, magnetic particles, for example, can be used for separation, which can bind to the cells via surface functionalization. Optionally, the separated cells can be washed to remove any remaining unwanted sample residue as completely as possible. For this purpose, a washing medium, for example a wash buffer, preferably located upstream in the apparatus, can be used, which, if a filter is used, can be rinsed through the filter along with the cells retained on it.

[0012] The separated cells are lysed in the device to release the nucleic acids contained within them. This can preferably be achieved using thermal lysis, in particular by heating the area of ​​the device where the separated cells are located.

[0013] The nucleic acids released from the separated cells by lysis are mixed with reagents for nucleic acid amplification and distributed between two amplification zones. The reagents may include, in particular, primers, polymerases, deoxyribonucleoside triphosphates (dNTPs), probes for (fluorescence) selection, buffer components, and / or other substances. Mixing with the reagents preferably occurs only after the nucleic acids have been divided into two parts, so that reagents specific to each amplification stage, especially primers, are added only to the respective part. Preferably, the reagents are arranged in the amplification zones, particularly freeze-dried as a water-soluble bead, where a bead is understood to be, in particular, a dried, especially freeze-dried, solid in spherical form commonly used in microfluidics.

[0014] Subsequently, nucleic acid fragments from both parts can be amplified in parallel in the amplification regions, particularly via polymerase chain reactions (PCR). For this purpose, the PCR reagents described above can be provided as a PCR master mix, especially in bead form. Parallel execution here means, in particular, that the amplifications in the two amplification regions are carried out at least partially simultaneously, preferably, but not necessarily, with the same start time. Specifically, the PCR cycles in the two amplification regions can proceed synchronously, at least temporarily.Preferably, each of the two amplification regions comprises at least two, preferably three, PCR chambers (PCR strand) connected in series via a fluidic system. These chambers can be heated to different temperatures to enable shuttle PCR, with the nucleic acids being shuttled between the different chambers at different temperatures according to the PCR cycles. Preferably, at least one chamber from each amplification region is located within the same temperature range. The temperature ranges preferably each encompass a portion of both amplification regions, with at least one chamber from each region.

[0015] The invention advantageously enables the rapid testing of a limited sample for multiple target sequences, and thus, in particular, for multiple candidates as causative agents of a disease. By separating the cells prior to lysis, a sufficient yield of amplifiable nucleic acids is advantageously achieved to ensure high sensitivity for each of the sequences in the amplification process, despite the parallel amplification of the multiple target sequences.

[0016] Preferably, PCR amplifications are performed as quantitative real-time PCR, which advantageously further accelerates the detection process. For example, the primers and detection probes in the reagents described above can be designed for the readout of multiple, for example, four fluorescence channels each, so that eight different target sequences, preferably one control sequence in each, and thus six pathogen sequences, can be amplified and detected in the two amplification regions. Particularly if the sample, especially due to its origin, does not have suitable candidates as control sequences for the amplifications, for example, nucleic acids from the patient's body cells (also called human control), control reagents, which specifically include control sequences, can be arranged in the apparatus.For example, the control reagents can be placed upstream of the device in a chamber, preferably freeze-dried as a bead. The control reagents may include, for example, plasmids, microorganisms, in particular bacteria, viruses, or fungi, and / or a protein shell containing DNA and / or RNA fragments as control sequences. The control reagents can be mixed with the sample or with the released nucleic acids before or after lysis, depending on the extent to which the lysis has beneficial and / or detrimental effects on the control reagents.

[0017] Lysing the separated cells preferably involves repeatedly conveying a fluid, in particular an elution medium such as an elution buffer, through the solid phase, preferably comprising repeated conveying in the same direction through the solid phase. If the solid phase includes a filter, the fluid can be moved, in particular pumped, through the same side of the filter through which the sample was moved to separate the cells. Thus, first the sample and then, preferably repeatedly, the fluid are moved into the filter through the same inlet and out of the filter through the same outlet. Due to the movement of the fluid, mechanical forces, in particular shear forces, are exerted on the cells between the fluid and the solid phase, which advantageously promote cell lysis.

[0018] According to an advantageous embodiment, chambers of the first amplification region and chambers of the second amplification region can be used for conveying the fluids during lysis. In particular, a PCR strand of one amplification region can be used to form a closed fluidic loop with the solid phase, through which the fluid can be repeatedly conveyed in the same direction over or through the solid phase. One or more chambers from the other amplification region can be used to temporarily hold a portion of the fluid. These chambers, serving as parking chambers, can be filled in one direction and emptied in the opposite direction, particularly through the same openings. Depending on the size of the chambers used and the amount of fluid used, the fluid can be divided into several fluid parts, which are also referred to as plugs.

[0019] The invention further relates to another microfluidic device, in particular as a further development of the microfluidic device described above.

[0020] The further apparatus comprises, in a first amplification section, first reagents for carrying out a first nucleic acid amplification. Preferably, the further apparatus comprises, in a second amplification section, second reagents for carrying out a second nucleic acid amplification. Nucleic acid amplification is understood to be a method for multiplying segments of nucleic acids, in particular a polymerase chain reaction (PCR) or isothermal amplification. Preferably, each of the two amplification sections comprises at least two, preferably three, PCR chambers (PCR chain) connected in series, which can be heated to different temperatures to enable the polymerase chain reactions to be carried out as shuttle PCR, wherein the nucleic acids are moved back and forth between the different chambers at different temperatures according to the PCR cycles.In the configuration with two amplification areas, preferably at least one chamber from each amplification area is located in the same temperature range. In this configuration, the temperature ranges preferably each encompass a portion of both amplification areas, with at least one chamber from each amplification area. The further device is specifically designed to perform parallel nucleic acid amplifications, particularly parallel PCR. Parallel performance here means, in particular, that the amplifications in the two amplification areas are carried out at least partially simultaneously, preferably, but not necessarily, with the same start time. In particular, the PCR cycles in the two amplification areas can run synchronously, at least temporarily.

[0021] Furthermore, the apparatus includes control reagents for monitoring the first and / or second nucleic acid amplification, in particular the PCR performed in each of the two amplification stages. The control reagents comprise, in particular, DNA and / or RNA segments, hereinafter also referred to as control sequences, which are preferably designed for at least partial amplification in the first and / or second nucleic acid amplification. As described above, this co-amplification serves to control the amplification process.

[0022] By arranging and, in particular, pre-storing the control reagents in the subsequent microfluidic apparatus, the control reagents, and especially the control sequences, can be directly provided and used for amplification, depending on the sample used. This is particularly useful when certain sample types, such as cerebrospinal fluid, inherently or with significant statistical variation, contain suitable control sequences. The control reagents can also contain primers for amplifying the control sequences. Alternatively or additionally, the first and / or second reagents can include primers for amplifying at least some of the control reagents. At least some of the control reagents, and in particular some of the control sequences, are preferably designed to react with the first and / or second reagents in the first and / or second nucleic acid amplification.In particular, the control sequences can be tailored to primers contained in the first and / or second reagents for amplification by using these primers. For example, the control reagents or the first and / or second reagents can contain buffer substances tailored to other substances in the first and / or second reagents or in the control reagents. The control reagents can be contained, at least partially, in a plasmid, in a microorganism, especially a bacterium, virus, or fungus, or in a protein shell. The control reagents can be present in liquid and / or solid form in the apparatus.For example, at least some of the control reagents are arranged in a preferably aqueous solution, in particular in a buffer, within the further apparatus. The solution, in particular the buffer, can be an elution medium or a binding medium, in particular an elution buffer or a binding buffer. Alternatively, the control reagents can be present in a separate solution or in a separate buffer.

[0023] Preferably, at least some of the control reagents are present in solid form, particularly in freeze-dried form, preferably as one or more preferably water-soluble beads, and preferably comprising the plasmids, microorganisms, and / or protein shells described above with the control sequences contained therein. A bead is understood to be, in particular, a dried, especially freeze-dried, solid in spherical form commonly used in microfluidics.

[0024] The first and / or second reagents described above are preferably also present as beads in separate bead chambers and comprise, for example, master mix reagents for PCR. For instance, these beads are beads comprising a PCR master mix each. A first bead chamber is preferably located in the first amplification region, and a second bead chamber is preferably located in the second amplification region.

[0025] A further presented method comprises first receiving a sample into a further microfluidic apparatus, wherein the sample comprises biological cells containing nucleic acids. The sample is prepared for amplification of at least portions of the nucleic acids contained in the sample, wherein the preparation may include lysis of cells contained in the sample to release the nucleic acids and / or purification of nucleic acids released from the sample on a solid phase, in particular a filter, of the further apparatus. Before or after preparation, the sample or the released nucleic acids may be mixed with control reagents located in the further apparatus. A first quantity of the released nucleic acids is then conveyed to a first amplification zone, and preferably a second quantity of the released nucleic acids to a second amplification zone.

[0026] Subsequently, a first amplification, in particular a PCR, of nucleic acid fragments from the first quantity can be carried out in the first amplification area, and preferably a second amplification, in particular a PCR, of nucleic acid fragments from the second quantity can be carried out in the second amplification area. Alternatively, only one

[0027] The amount of released nucleic acids is transported to only one of the two amplification regions and subjected to amplification there.

[0028] Brief description of the drawings

[0029] Exemplary embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. The same reference numerals are used for the elements shown in the various figures that have a similar effect, thus avoiding a repeated description of the elements.

[0030] They show

[0031] Figure 1 shows a flowchart of an embodiment of the method according to the invention.

[0032] Figure 2 shows an embodiment of the microfluidic device according to the invention and

[0033] Figures 3a-c show schematic snapshots of the microfluidic device shown in Figure 2 at three points in time during the execution of the embodiment of the method according to the invention.

[0034] Embodiments of the invention

[0035] Figure 1 shows a flowchart for an embodiment of the method 500 according to the invention, which is described below and can be carried out in particular with an embodiment of the microfluidic device 100 according to the invention, shown schematically in Figure 2. For example, the method 500 is used to detect bacteria in a sample from a mammal, in particular a human. The sample contains a body fluid in which these bacteria are suspected to be present. For example, the sample contains cerebrospinal fluid (CSF) from a lumbar puncture for the detection of pathogens for bacterial meningitis, preferably with only one sample, six pathogens are to be detected, namely Escherichia coli, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, and Haemophilus influenzae.After collection, the sample can be transferred to a transport medium from the organism to the microfluidic device 100. The transport medium preferably comprises a cell-stabilizing medium without lysing properties, such as Copan Universal Transport Medium®, Amies medium, or weak saline solutions based on sodium chloride or phosphate-buffered saline (PBS).

[0036] The microfluidic device 100 is, in particular, a microfluidic cartridge 100 which can be processed with an analytical instrument (not shown), as described, for example, in documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1, without fundamentally limiting the invention thereto. The schematic representation of the microfluidic device 100 in Figure 2 shows, in particular, but not exhaustively, some exemplary chambers of the device, wherein these chambers are partially connected to one another by fluidic channels and can also be temporarily separated from one another by valves (not shown), for example, diaphragm valves.The cartridge can have a multi-layered structure, for example, made of polycarbonate, in particular a fluidic layer containing the chambers and channels for sample processing, and a pneumatic layer with channels and chambers as well as pneumatic connections to the analyzer. By applying negative or positive pressure to the pneumatic connections, a membrane located between the pneumatic and fluidic layers, for example comprising thermoplastic polyurethane (TPU), can be moved partially within recesses, in particular chambers or channels, of the fluidic and pneumatic layers to implement the functions of pump chambers and diaphragm valves.

[0037] The device 100 can, as shown in Figure 2, for example, have two PCR strands in two amplification regions 101, 102, each PCR strand comprising several fluidically connected chambers arranged in series. As shown, each PCR strand can have three fluidically connected chambers 111, 112, 113, 121, 122, 123 in order to perform, in particular, two independent PCR reactions with three temperature ranges 161, 162, 163 (for example, for each strand in the sequence 92 to 99 °C for denaturation, 70–75 °C for elongation, and 60–65 °C for primer hybridization, corresponding to one PCR cycle) in parallel as shuttle PCRs.The amplification areas 101 and 102 can include further chambers, such as a parking chamber 120 in the second amplification area 102, which, as shown in Figure 2, is fluidically upstream of the first PCR chamber 121 in the second PCR strand. For example, these chambers 111, 112, 113, 120, 121, 122, and 123 each have a volume of 20 pl. However, the method is not limited to such a specific architecture.

[0038] The device 100 comprises, in the first amplification area 101, first reagents 141 for carrying out a first nucleic acid amplification and, in the second amplification area 102, second reagents 142 for carrying out a second nucleic acid amplification. These reagents 141, 142 can, in particular, comprise primers, deoxyribonucleoside triphosphates (dNTPs), auxiliary oligonucleotides, and enzymes, especially a polymerase, and / or buffer reagents for carrying out the nucleic acid amplification. This embodiment describes the invention for carrying out two parallel polymerase chain reactions (PCRs) distributed across the two PCR strands, such that the first and second reagents can, in particular, be PCR master mix reagents 141, 142, which are preferably freeze-dried.

[0039] As shown in Figure 2, the reagents 141, 142 are preferably each in the form of a bead 141, 142 in a first bead chamber 114 and in a second bead chamber 124, respectively. The first bead chamber 114 is arranged in the first amplification region 101 and is directly connected to the first chamber 111 and the last chamber 113 of the first PCR strand 111, 112, 113, while the second bead chamber 124 is arranged in the second amplification region 102 and is directly connected to the last chamber 123 of the second PCR strand 121, 122, 123 and, via the parking chamber 120, to the first chamber 121 of the second PCR strand 121, 122, 123. This minimizes transport times, transport distances and fluid losses when transporting fluid between bead chamber 114, 124 and the directly connected PCR strand.

[0040] In particular, if the sample does not contain a sufficient number of sequences suitable for monitoring the amplification, especially a human control, the device 100 may further comprise control reagents 151 for monitoring the first and / or second nucleic acid amplification, especially the PCR in the two PCR strands. The control reagents comprise, in particular, nucleic acid sequences, especially DNA sequences, hereinafter referred to as control sequences, which are amplified along with the nucleic acid amplifications and on the basis of whose amplification the quality of the reaction can be assessed, especially in real time. In particular, these control sequences are naturally occurring sequences, for example, sequences found in mammals, or alternatively, artificial sequences.The control reagents 151, or alternatively the first or second reagents 141, 142, preferably also include primers matched to the control sequences so that the nucleic acid segments corresponding to the control sequences can be amplified during amplification. The control reagents 151 can also include fluorescent probes matched to the control sequences as control probes, particularly to monitor the amplification during a real-time polymerase chain reaction.

[0041] The control reagents can be arranged in the device 100, particularly in solid, especially lyophilized, form, preferably as a bead, or in liquid form. In the case of liquid pre-storage, the control reagents can be pre-stored as a component of a liquid reagent, particularly as part of a buffer, in a reagent chamber 131, 132, 133, either as a separate reagent or buffer, or mixed with a reagent or buffer already intended for another function, for example, in a lysis buffer or binding buffer. In the case of solid pre-storage, particularly as a bead 151, the control reagents can be pre-stored in a separate control bead chamber 150, as shown in Figure 2 of this example.The control sequences, in particular as DNA and / or RNA fragments, i.e., as oligo- or polynucleotides, can be arranged in the device 100 in various ways, in liquid or solid form, in particular as Bead 151, as required. For example, at least some of the DNA and / or RNA fragments of the control sequences can be contained in a vehicle such as a plasmid, a microorganism, in particular a bacterium, virus, or fungus, or a protein coat as so-called armored RNA / DNA, which in turn can be present in the buffer or freeze-dried in Bead 151. Such control sequences are commercially available, for example, Armored DNA Quant™, or can be created specifically for the respective application.

[0042] The control bead chamber 150 is fluidically connected to a sample input chamber 105 of the device 100, so that a sample taken into the device via the sample input chamber 105 in a first step 501 of the method 500 can be mixed directly with the control reagents 151 if the addition of the control reagents is necessary, particularly due to a lack of human control in the sample. For example, in a second step 502 of the method 500, the sample can be conveyed to the control bead chamber 150 to dissolve the bead 151. In an alternative second step 502, a liquid, for example a buffer, can be introduced from a reagent chamber 132 connected to the control bead chamber 150 into the control bead chamber 150 to dissolve the bead 151 and then mix it with the sample, for example in the sample input chamber 105.

[0043] The sample can then be prepared for the amplification of nucleic acids contained in the sample. This preparation, in a third step 503 of the process 500, initially includes the purification of cells contained in the sample, in particular the bacteria to be detected. For this purpose, the sample is rinsed through a porous filter 107 in a filter chamber 106 of the apparatus 100, in particular by means of a membrane pump of the apparatus, which retains these cells and thus separates them from other sample components. This is also known as the cell capture method. The control sequences, which may also be contained in the sample, can also be retained by selecting the appropriate size of the filter 107 and / or by non-specific binding to the filter material, especially if they are located in a plasmid or a microorganism as described above.

[0044] In a fourth step 504, the retained cells and optionally the vehicles of the control sequences are lysed, for example by thermal lysis. For this purpose, the first temperature range 161, in which the filter chamber 106 is located, can be heated, for example, to 60 °C. Preferably, however, the filter of the filter chamber 161 is also used for the lysis. For this purpose, an upstream fluid, in particular a buffer such as an elution buffer, can be used, which, after separation of the cells on the filter 107, is preferably conveyed, in particular pumped, several times over the filter, and thus, together with the filter, exerts additional mechanical forces, in particular shear forces, on the cells, which enhances the lysis effect. The fluid is moved onto the filter from the side on which the cells were retained.In particular, the filter 107 with the cells accumulated on it can first be heated before the fluid 134, which is stored, for example, in a reagent chamber 133 (indicated by diagonal hatching in Figure 2), is added. Advantageously, the second PCR strand in the second amplification chamber 102 can be used to repeatedly circulate the fluid 134 in a loop, for example, between 10 and 20 times, over the filter 107, starting, for example, from the filter chamber 106, through the third PCR chamber 123, the second PCR chamber 122, the first PCR chamber 121, and the parking chamber 120, before being reconnected to the filter chamber 106 in that order. The amount of fluid used for lysis can be selected depending on the requirements of the subsequent procedure.If more fluid is required, for example between 50 and 100 microliters, than can fit into filter chamber 106, the fluid can be divided into several parts, hereinafter referred to as plugs, according to chamber sizes 106, 120, 121, 122, 123, and successively pumped through filter 107. For example, with 80 microliters of fluid and a PCR chamber size of 20 microliters (including parking chamber 120), four of these chambers would always be filled with fluid. To facilitate the fluid flow, one or more chambers 111, 112, 113 of the first PCR strand in the first amplification area 101 can also be advantageously used. For example, up to three plugs passed through filter 107 can initially be parked in the first PCR strand 111, 112, 113, while the remaining plugs are pumped through filter 107 and introduced into the second PCR strand. Figure 3a illustrates this by diagonal hatching of the chambers.

[0045] 113. 112. 111. 120 schematically a snapshot at the time when three plugs are parked in the three PCR chambers 111 , 112 , 113 of the first PCR strand and the fourth plug is still in the parking chamber 120 in the second amplification area 102 (the volume of the filter chamber 106 is neglected here due to its comparatively small size). Starting from the situation in Figure 3a, the plug can first be moved from the parking chamber 120 via the filter chamber 106 including filter 107 and via the second PCR strand 123, 122, 121 back into the parking chamber 120, before the plugs parked in the first PCR strand are pumped out of the first PCR strand 113, 112, 111, so to speak in reverse, and transported into the second PCR strand for further transport via the filter 107.Alternatively, the plug can remain in parking chamber 120, while the other plugs are transferred to the second PCR strand, optionally after temporary parking in the first PCR strand, particularly for fluid mechanics reasons. Figure 3b illustrates this by diagonal hatching of the chambers.

[0046] Figures 123, 122, 121, and 120 schematically represent a snapshot at the point when the three plugs are again in the three PCR chambers 123, 122, and 121 of the second PCR strand, as well as in the parking chamber 120, before they are passed over filter 107 again. The plug in parking chamber 120 is then transferred, together with the plugs from the first and second PCR chambers 121 and 122 of the second strand, into the three PCR chambers 111, 112, and 113 of the first strand, so that the plug from the third PCR chamber 123 now takes the place of the plug in the parking chamber shown in Figure 3a in the second strand, thus changing the order of the plugs. Alternatively, instead of three plugs, only two plugs per cycle could be temporarily parked in the first PCR strand.The temporary deactivation of at least some of the plugs in the first PCR strand also has the advantage that the resulting change in the order of the plugs with each cycle leads to better mixing of all plugs with the lysed nucleic acids. During lysis 504, the fluid 134 is thus conveyed in a combined process comprising a loop-shaped movement over the second PCR strand and the filter chamber 106, and a back-and-forth movement in the first PCR strand repeatedly over the filter 107. This process can be carried out, for example, 10 to 20 times, as already mentioned, which requires a time span of, for example, one to two minutes.

[0047] Thus, after the end of lysis, for example 504, there are four plugs of the fluid containing released nucleic acids.

[0048] In a fifth step 505, the purified nucleic acids can be divided between the two amplification chambers 101 and 102, for example, two plugs for each amplification chamber 101 and 102, and first mixed with the PCR master mix bead 141 and 142 in the first bead chamber 114 and second bead chamber 124, respectively, to dissolve the beads 141 and 142. Figure 3c schematically shows, by means of oblique hatching of chambers 111, 114, 120, and 124, a snapshot at the time when one plug each is located in the first and second bead chambers 114 and 124, one plug is located in the third PCR chamber 113 in the first amplification chamber 101, and one plug is located in the third PCR chamber 123 in the second amplification chamber 102.The PCR mastermix beads 141, 142 are still shown, so that the representation in Figure 3c is a point in time shortly after the insertion of the plugs into the bead chamber 114, 124 and before complete dissolution of the PCR mastermix beads 141.

[0049] Subsequently, in a sixth step, amplification can take place in the PCR strands, particularly parallel amplifications, by introducing at least the plug dissolved in the PCR master mix beads 141, 142 into the PCR strands for each amplification region. For example, this involves quantitative or qualitative real-time polymerase chain reactions, which can be read out in real time via fluorescence radiation. The fluorescence radiation also includes radiation in a wavelength range specified by the control probes for monitoring the amplifications. By using both PCR strands, several target sequences, for example six, can be amplified in parallel alongside two control sequences in the qPCR method and read out via disjoint fluorescence channels.Alternatively, the first and / or PCR strand could have only two chambers instead of three for mapping and carrying out the PCR cycle, so that, for example, the second chambers 112, 122 are intended for denaturation and the third chambers 113, 123 are used for both annealing and elongation, and the first chambers are omitted in this configuration.

[0050] Similarly, chambers 120, 112, 113, 122, 123 could be used for the shuttle lysis described above in step 4 504.

Claims

Claims 1. Method (500) for carrying out parallel nucleic acid amplifications, in particular parallel polymerase chain reactions, with a microfluidic device (100), comprising the steps: • Intake (501) of a sample into the microfluidic device (100), wherein the sample comprises biological cells with nucleic acids contained in the cells. • Separation (502) of at least some of the cells from the sample, in particular by size exclusion, with a solid phase (107), for example via a filter (107). • Lysing (503) the separated cells to release nucleic acids located in the separated cells • Transporting (504) a first quantity of the released nucleic acids to a first amplification area (101) and a second quantity of the released nucleic acids to a second amplification area (101). • Performing (505) a first amplification, in particular a PCR, of nucleic acid fragments of the nucleic acids from the first quantity in the first amplification area (101) and a second amplification, in particular a PCR, of nucleic acid fragments of the nucleic acids from the second quantity in the second amplification area (102).

2. Method (500) according to claim 1, wherein the lyse (503) is carried out by heating the separated cells, in particular by heating a region comprising the solid phase (107).

3. Method (500) according to one of the preceding claims, wherein the lyse (503) comprises preferably multiple conveying of a fluid (134), in particular an elution medium (134), over the solid phase (107) after separation of the cells, preferably comprising repeated conveying in the same direction through the solid phase (107).

4. Method (500) according to claim 3, wherein chambers (111 , 112, 113) of the first amplification area (101) and chambers (120, 121 , 122, 123) of the second amplification area (102) are used for conveying the fluid during lysis, in particular for loop conveying over the second PCR strand (120, 121 , 122, 123) and / or forward and backward movement in the first PCR strand (111 , 112, 113).

5. Method (500) according to claim 3 or 4, wherein the fluid (134) is divided into several fluid parts.

6. Method (500) according to one of the preceding claims, wherein after lyse (503) the separated cells are washed with a washing medium, in particular by rinsing the washing medium over the solid phase (107).

7. Method (500) according to any of the preceding claims, wherein the first quantity of released cells is mixed with first reagents (141) for the first amplification and the second quantity of released cells is mixed with second reagents (142) for the second amplification.

8. Method (500) according to one of the preceding claims, wherein the first reagents (141) are placed upstream in the first amplification area (101), in particular as a bead (141), and the second reagents (142) are placed upstream in the second amplification area (102), in particular as a bead (1042).

9. Method (500) according to any of the preceding claims, wherein the sample is mixed with control reagents (151) after uptake (501) for control of the first and / or second nucleic acid amplification.

10. Method (500) according to claim 9, wherein the control reagents (151) are placed upstream in the device (100), in particular as a bead (151).

11. Method (500) according to any of the preceding claims, wherein the amplifications are carried out as quantitative real-time polymerase chain reactions (505).

12. Microfluidic device (100), in particular a microfluidic cartridge, for carrying out parallel nucleic acid amplifications, in particular parallel polymerase chain reactions, wherein the device (100) has in a first amplification area (101) first reagents (141) for carrying out a first nucleic acid amplification and preferably in a second amplification area (102) second reagents (142) for carrying out a second nucleic acid amplification and wherein the device (100) has control reagents (151) for a control of the first and / or second nucleic acid amplification.

13. Microfluidic device (100) according to claim 12, wherein the control reagents (151) comprise DNA segments and / or RNA segments which are preferably designed for at least partial amplification in the first and / or second nucleic acid amplification.

14. Microfluidic device (100) according to claim 12 or 13, wherein at least some of the control reagents (151) are designed to be compatible with the first and / or second reagents (141, 142) for a reaction in the first and / or second nucleic acid amplification.

15. Microfluidic device (100) according to claim 12, 13 or 14, wherein at least some of the DNA segments and / or RNA segments are contained in a plasmid, a microorganism, in particular in a bacterium, virus or fungus, and / or are surrounded by or embedded in a protein shell, wherein preferably at least some of the control reagents (151) are contained in one or more beads (151) or in a preferably aqueous solution, in particular in a buffer, in which they are arranged in the device.

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