Method for carrying out nucleic acid amplification using a microfluidic device

Abstract

The invention relates to a method (500) for carrying out nucleic acid amplification, in particular an isothermal nucleic acid amplification, using a microfluidic device (100), in particular using a PCR architecture (101, 102).

Description

[0001] Description

[0002] title

[0003] METHOD FOR PERFORMING NUCLEAN ACID AMPLIFICATION USING A MICROFLUIDED DEVICE

[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 implemented at the point of care and as immediate, near-patient diagnostics using microfluidic systems. In this process, a patient sample is introduced into a microfluidic cartridge, which is then activated within an analyzer to perform nucleic acid amplification inside the cartridge.

[0006] Methods used for nucleic acid amplification include, in particular, the polymerase chain reaction with its two- or three-stage temperature cycling, or isothermal amplification reactions with nucleic acid amplification at only one temperature level. Transferring the workflows of such fundamentally known methods from the macroscopic laboratory to an integrated microfluidic system requires an innovative implementation. Disclosure of the invention

[0007] Advantages of the invention

[0008] Against this background, the invention relates to a method for carrying out nucleic acid amplification with a microfluidic device and to a device configured for the method. Such a device 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.

[0009] Nucleic acid amplification is a method for multiplying sections of nucleic acids, in particular a polymerase chain reaction (PCR) or isothermal amplification.

[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 body tissue, for example, blood, sputum, 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 is placed in a medium for transport from the living organism to the analysis, i.e., to the microfluidic device. This transport medium, for example, Copan eNAT™, may contain lyses that lyse the biological cells contained in the sample, thereby releasing the nucleic acids within the cells.After sampling, the sample can preferably first be purified, wherein the purification particularly comprises the purification of the nucleic acids contained in the sample and intended for amplification, preferably the purification of the nucleic acid via a solid phase of the apparatus. In this process, the nucleic acids are separated from the rest of the sample by binding to the solid phase, in particular to a filter, and subsequently, preferably after removal of sample residues with a washing medium, dissolved again from the solid phase to form a portion of the purified sample, particularly with an elution buffer used for dissolution.

[0012] The sample is then mixed with reagents specific for nucleic acid amplification, and the resulting mixture is hereinafter referred to as the first mixed sample. The specific reagents are, in particular, reagents that are specific for amplifying the one or more nucleic acid segments to be amplified, for example, primers or auxiliary primers. Depending on the nucleic acid segment to be amplified, these reagents, especially the primers, may differ. The specific reagents are preferably placed upstream in the microfluidic apparatus and may be present in dried, in particular lyophilized, form before mixing, especially as part of a bead, which is hereinafter referred to as the detection-specific bead or primer bead.A bead is understood to be, in particular, a dried, especially freeze-dried, solid in spherical form, commonly used in microfluidics. Alternatively, the specific reagents can also be present in liquid form, for example, in an aqueous solution. The specific reagents can be stored in a chamber, hereinafter referred to as the first bead chamber. Depending on the application, the primer bead can also include additional non-specific reagents. The rehydration of the dried specific reagents is preferably carried out by mixing them with the sample. To dilute the reagents or the sample, before or after rehydration of the reagents, or alternatively to rehydration by the sample, further liquid, in particular a buffer such as an elution buffer placed upstream in the apparatus, can be added.

[0013] The nucleic acids contained in the first mixed sample are subsequently denatured. These nucleic acids can be, in particular, oligonucleotides contained in the added specific reagents, especially primers, and / or nucleic acids from the sample itself, i.e., nucleic acids from the organism. The denaturation can be carried out by heating at least a portion of the first mixed sample to a temperature higher than 90 °C, preferably higher than 92 °C, and most preferably higher than 95 °C, for example, 98 °C. Due to the denaturation process, preferably only those reagents are included in the detection-specific bead that are not substantially affected or damaged by the denaturation, particularly by heating, or for which a partial loss is acceptable.The denaturation takes place in a denaturation area of ​​the microfluidic device, preferably comprising one or more denaturation chambers.

[0014] According to a particular embodiment, the denaturation section forms part of a first PCR section of the device, i.e., a section, in particular comprising chambers, for carrying out PCR. Advantageously, one or more chambers of the PCR section can be used for denaturation according to the method, i.e., as denaturation chambers. In the case of isothermal amplification, a PCR architecture of the microfluidic device, in particular a PCR strand, can thus be advantageously used, in a sense, for the denaturation of nucleic acids contained in the first mixed sample. A PCR strand is understood to mean, in particular, two or three fluidically connected chambers for carrying out shuttle PCR, i.e., a PCR in which the sample to be amplified is shuttled between the two or three chambers.Three chambers, each at a different temperature according to a PCR cycle, are repeatedly moved back and forth. Denaturation can take place, in particular, in one or more chambers of a first PCR strand of the microfluidic device, with one of these chambers preferably being designated for denaturation during PCR, for example, by means of a heater in the analyzer to heat this chamber to a denaturation temperature of, for example, 99 °C. According to a preferred embodiment, the first bead chamber described above can be arranged in the first PCR section and preferably be directly fluidically connected to at least one of the chambers of the PCR strand of the first PCR section via a channel, for example, separable via a valve.

[0015] Following denaturation, the denatured sample is mixed with reagents relevant to the specific type of nucleic acid amplification. These reagents are hereinafter referred to as process-specific reagents, and the resulting mixture is referred to as the second mixed sample. The process-specific reagents are primarily those required for nucleic acid amplification. These reagents are preferably independent of the choice of nucleic acid segments to be amplified and may differ depending on the type of nucleic acid amplification. For example, the process-specific reagents may include reagents commonly used for the respective reaction or amplification method, such as polymerases, deoxyribonucleoside triphosphates (dNTPs), probes, reaction buffers, other enzymes, or additives.If the method involves isothermal amplification, the method-specific reagents may include, for example, a strand-displacing polymerase, auxiliary enzymes such as reverse transcriptases, endonucleases such as restriction or nicking enzymes, helicases, recombinases, single-strand binding proteins, ligases, or other substances. If, on the other hand, a classical PCR is performed, a Taq polymerase, for example, may be used as the method-specific reagent. Due to their heat resistance, Taq polymerase or other thermostable polymerases may alternatively be included as part of the specific reagents described above, and particularly in the detection-specific bead.

[0016] The process-specific reagents can be present in dried, particularly lyophilized, form prior to mixing, especially as part of a bead, which is hereinafter referred to as a process-specific bead or enzyme bead. Alternatively, the process-specific reagents can also be present in liquid form, for example, in aqueous solution. The process-specific reagents can be located in a chamber, hereinafter referred to as the second bead chamber. According to a preferred embodiment, the process-specific reagents are arranged in a second PCR region of the apparatus. In particular, the second bead chamber can be located within the second PCR region. The second bead chamber can preferably be directly connected fluidically to at least one of the chambers of a PCR strand of the second PCR region via a channel, for example, via a valve.The specific reagents and the process-specific reagents are thus arranged separately in or upstream of the microfluidic device, in particular the specific reagents as primer beads in the first bead chamber and the process-specific reagents as enzyme beads in the second bead chamber. Depending on the application, the enzyme bead may also include additional reagents. The rehydration of the dried process-specific reagents is preferably carried out by mixing them with the denatured sample. For dilution of the reagents or the sample, before or after rehydration of the reagents, or alternatively to rehydration by the sample, additional liquid, in particular a buffer such as an elution buffer upstream of the device, can be added.

[0017] The local separation of specific reagents and process-specific reagents advantageously also supports thermal separation of their upstream storage locations. Thus, the respective processing of these reagents—i.e., denaturation or amplification reactions, particularly after rehydration—can take place directly at or near, especially adjacent to, the upstream storage location. This advantageously reduces or completely eliminates transport times and losses between mixing with the reagents and processing.In a preferred embodiment, the denaturation area, in particular the denaturation chamber, and the process-specific reagents, in particular the second bead chamber, are arranged in the apparatus such that heating the denaturation area from a first initial temperature to a denaturation temperature during a denaturation period does not raise the temperature of the process-specific reagents, in particular the second bead chamber, above a maximum temperature from a second initial temperature. This has the advantage that the process-specific reagents are not affected, or only minimally affected, by the heating during denaturation. The denaturation temperature is in particular 92 °C, more preferably 95 °C, and most preferably 99 °C, with the denaturation period being 2 minutes, more preferably 5 minutes, and most preferably 10 minutes.The maximum temperature can be, for example, 70 °C, preferably 60 °C, most preferably 50 °C, while the first and / or the second output temperature can be, for example, 20 °C, preferably 30 °C, most preferably 40 °C.

[0018] The second mixed sample, or a part thereof, can then be used to carry out nucleic acid amplification in an amplification area, in particular in at least one amplification chamber, of the microfluidic device.

[0019] Nucleic acid amplification can, in particular, comprise isothermal amplification of nucleic acid fragments contained in the second mixed sample. According to a preferred embodiment, an existing PCR architecture of the microfluidic device can advantageously be repurposed for isothermal amplification. In particular, one or more chambers of a PCR strand can serve as amplification chambers, especially an architecture for, in particular quantitative, real-time PCR with real-time readout of one of the PCR chambers, for example, via fluorescence radiation.When using multiple amplification chambers, at least a portion of the second mixed sample can be repeatedly moved back and forth between a first and a second amplification chamber of the device, particularly to enable or perform optical selection of the portion from the second amplification chamber during amplification. This is especially advantageous if the first chamber can be heated from several sides, particularly from two opposite sides, while the second chamber is optically visible for selection from at least one side, particularly through a transparent wall of the chamber.

[0020] Brief description of the drawings

[0021] 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.

[0022] They show

[0023] Figure 1 shows a flowchart of an embodiment of the method according to the invention.

[0024] Figure 2 shows an embodiment of the microfluidic device according to the invention and

[0025] 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.

[0026] Embodiments of the invention

[0027] Figure 1 shows a flowchart for a subsequently described embodiment of the method 500 according to the invention, which can be carried out in particular with an embodiment of the microfluidic device 100 according to the invention shown schematically in Figure 2.

[0028] 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 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.

[0029] In a first step 501, the sample, for example comprising 300 microliters (pl), is drawn into an input chamber 105 of the cartridge 100. The sample is a biological sample containing nucleic acids, for example a swab or body fluid such as blood or sputum from a mammal, particularly a human. The sample is already mixed with a lyse medium, for example COPAN eNAT™, which lyses the cells in the sample and thereby releases the nucleic acids contained within the cells.

[0030] In a second step 502, the sample is purified. For even more thorough lysis, the sample can first undergo a further lysis step, for example, using ultrasound. For this purpose, the analyzer preferably has an ultrasound source located near the input chamber 105 of the cartridge housed in the analyzer. During the purification process, for example according to the known bind-wash-elute method, the sample can be mixed with a binding buffer, which is preferably located upstream of the cartridge, for example in a reagent bar in a first reagent chamber 131, so that during subsequent rinsing of the sample through a filter (not shown) in a filter chamber 106, the nucleic acids bind to the filter.After washing the bound nucleic acids, for example with a washing buffer placed upstream in a second reagent chamber 132, the nucleic acids are released from the filter again with an elution buffer preferably placed upstream in a third reagent chamber 133. This eluate, which may comprise, for example, 60 pl, is hereinafter also referred to as the purified sample. A first portion of this eluate, which was the first to pass through the filter, for example 20 pl, can be discarded and thus disregarded for the process, since this first portion may still contain the largest proportion of washing buffer retained by the filter.

[0031] The cartridge can have two PCR strands in two PCR regions 101, 102, each PCR strand comprising several fluidically arranged chambers in series. As shown in Figure 2, each PCR strand can have three chambers 111, 112, 113, 121, 122, 123 connected in series, in particular to perform two independent PCR reactions, each with three temperature ranges. For example, these chambers 111, 112, 113, 121, 122, 123 each have a volume of 20 pl. However, the method is not limited to such an architecture, especially when performing isothermal amplification. Conversely, when performing isothermal amplification, a PCR architecture of the microfluidic cartridge can be used for a different purpose.In particular, two PCR areas 101 , 102 separated from each other can be advantageously used for the procedure, especially, as described below by way of example, the first PCR area 101 for the pre-storage of the specific reagents 141 and for the denaturation, and the second PCR area 102 for the pre-storage of the procedure-specific reagents 142 and for the amplification reaction as an amplification area.

[0032] In a third step 503 of the procedure 500, for example, 40 pl of the purified sample are first divided equally between two of the chambers 112 and 113 of the first PCR strand. Subsequently, for example, 30 pl of the 40 pl eluate is transferred to a first bead chamber 114 connected to the first PCR strand. This first bead chamber 114 contains primers as specific reagents 141 for nucleic acid amplification. In this example, these primers 141 are present in the first bead chamber 114 as a lyophilized bead. The bead, also referred to as the primer bead, is rehydrated and dissolved by the purified sample, thereby mixing the primers with the purified sample. The resulting mixture is hereinafter referred to as the first mixed sample.As shown in Figure 2, the first bead chamber 114 is arranged in the first PCR area 101 and directly connected to the first PCR strand 111, 112, 113, so that transport times, transport distances and fluid losses during fluid transport between the first bead chamber 114 and the first PCR strand are kept to a minimum. Alternatively, the first bead chamber 114 could also be arranged at another location in the device 100.

[0033] At least a portion of the first mixed sample, for example 20 pl, is returned to one or two chambers 112, 113 of the first PCR strand to denature the nucleic acids and preferably also the primers contained therein in the fourth step 504 of the process 500 by heating to above 90 °C, for example to 98 °C for a duration of, for example, 60 seconds. This portion of the first mixed sample can be mixed before or after denaturation with, for example, another 20 pl of elution buffer from the third reagent chamber 133, in particular to provide sufficient volume, if necessary, for dissolving the process-specific reagents 142 as described below in the fifth step 505. In the present example, this dilution of the portion takes place before denaturation, so that the diluted portion is distributed into two chambers 111, 112 of the first PCR strand and denatured there.Figure 3a schematically shows a snapshot of the denaturation of the device 100 shown in Figure 2. The interior of the two chambers 111, 112 is filled with a slanted line pattern to illustrate the filling of these chambers 111, 112 with part of the first mixed sample. Both chambers 111, 112 are located in a heating zone 151, 152, for example, by contacting the outside of the device 100 in these heating zones 151, 152 with heaters of the analyzer. The device may have further heating zones not shown. For example, the first heating zone 151, which comprises the first chamber 111 of the first PCR strand, is provided and configured for the denaturation step during the PCR cycle, for example, for heating to 99 °C.For example, the second heating area 152, which comprises the second, i.e., here middle, chamber 112 of the first PCR strand, is provided and configured for the elongation step during the PCR cycle, for example, for heating to 70 °C, wherein this heating area 152, and in particular the corresponding heater of the analyzer, is preferably also capable of heating to a denaturation temperature such as 99 °C. Alternatively, the first PCR strand could be designed to use only two instead of three chambers to perform the PCR cycle, so that, for example, the second chamber 112 is provided for denaturation and the third chamber 113 for both annealing and elongation, and thus the second chamber 112 would also be suitable for performing the denaturation 504 for the presented method 500.The two heating zones 151 and 152 can therefore also be considered the denaturation zone described above. If dilution only takes place after denaturation, the denaturation and thus the heating can only occur in one of the chambers 111 and 112, in particular in the first chamber 111 of the first PCR strand in the first heating zone 151.

[0034] The denatured portion, hereinafter referred to as the denatured sample, or at least a portion thereof, for example 20 pl, is transferred in the fifth step 505 to a second bead chamber 124. This second bead chamber 124 contains enzymes and other reagents, specifically process-specific reagents 142 for nucleic acid amplification, in particular a polymerase, deoxyribonucleoside triphosphates (dNTPs), and / or auxiliary enzymes for isothermal amplification. In this example, these substances 142 are present in the second bead chamber 124 as a lyophilized second bead 142. The bead, also referred to as the enzyme bead, is rehydrated and dissolved by the purified sample, thereby mixing the enzymes with at least the portion of the denatured sample. The resulting mixture is hereinafter referred to as the second mixed sample.Figure 3b schematically shows a snapshot of this process, with the interior of bead chamber 124 filled with a slanted line pattern to indicate that the bead chamber has already been filled with the portion of the denatured sample. While the primer bead has already dissolved and is therefore no longer shown in Figure 3b, the enzyme bead 142 is still depicted to illustrate that its dissolution is just beginning. The enzyme bead can also contain probes for optical readout during or after nucleic acid amplification. Alternatively, such probes can also be contained in the primer bead if the high temperature during denaturation is not a problem for these probes.The second bead chamber 124 can be arranged in the second PCR section 102, as shown in Figures 2 and 3b, and directly connected to chambers 121, 122, and 123 of the second PCR strand. This allows a larger quantity, for example, 30 pl, of the denatured sample to be conveyed towards the second PCR strand. A portion of this sample, for example, the 20 pl already described, can be directed into the second bead chamber 124, while the remainder, for example, for later remixing, can be directed into chamber 123 of the second PCR strand and stored there. Alternatively, the second bead chamber 124 could also be arranged in the first PCR section 101 or at another location within the apparatus 100.The arrangement of the enzyme bead 142 in a chamber 124 at a distance from the chambers 112, 113 of the first PCR area 102, in which the denaturation is carried out, has the advantage that the enzyme bead is spared as much as possible from the heat effect of the denaturation. Taking into account the thermal conductivity of the cartridge material, for example polycarbonate, and the geometric shape of the cartridge area between the denaturation chambers 112, 113 and the second bead chamber 124, the distance between the chambers 112, 113, 124 can be chosen in the cartridge design such that a heating from, for example, 40°C to a minimum denaturation temperature of, for example, 92°C in the area of ​​the denaturation chambers 112, 113 for a minimum time of, for example, one or two minutes does not heat the second bead chamber 124 from, for example, 40°C to more than 50°C.

[0035] In a sixth step 506 of the process 500, the second mixed sample can then be amplified. In particular, the purified nucleic acids contained in the second mixed sample can be amplified together with the denatured primers also present therein and enzymes dissolved from the second bead 142. For example, the second mixed sample can be transferred to a chamber 123 of the second PCR strand for isothermal amplification and heated there to the desired temperature level, for example, 63 °C, for the duration of the amplification. This is schematically illustrated as a snapshot in Figure 3c, again with a slanted line pattern, this time showing the amplification chamber 123 filled with the second mixed sample.The amplification chamber 123, which, as shown, forms the third (here, lowest) chamber 123 of the second PCR strand, is preferably located in a third heating zone 153 of the microfluidic device 100. For example, this third chamber 123 is intended for the primer hybridization step, also called the primer annealing step, during the PCR cycle and is configured to be heated to a temperature between 60 and 65 °C, for example by a heater of the analyzer that contacts the device in the third heating zone 153. Since both the second bead chamber 124 and the amplification chamber 123 are located in the second PCR zone, transport times, transport distances, and fluid losses during the transport of the second fluidic sample can also be kept as low as possible.Alternatively or additionally, another chamber can be used for amplification, in particular the middle chamber 122 of the second PCR strand, for example, which is intended for elongation or alternatively denaturation. During isothermal amplification, the second mixed sample to be amplified can also be moved back and forth between two chambers 122, 123 as the first and second amplification chambers 122, 123, for example, if the first amplification chamber 122 can be heated from two sides, while the second amplification chamber 123 can only be heated from one side, in order to allow optical readout of the second amplification chamber 123 from the other side.In the case of isothermal amplification, the reaction can be monitored at regular intervals, for example every 30 seconds, via fluorescence signals and terminated when a predetermined threshold of fluorescence radiation in a specific wavelength range is exceeded.

Claims

Claims 1. Method (500) for carrying out nucleic acid amplification, in particular isothermal nucleic acid amplification, using a microfluidic device (100), comprising the steps: • Intake (501) of a sample into the microfluidic device (100) • Mixing (503) the preferably purified sample with reagents (141) specific for nucleic acid amplification, in particular primers, to obtain a first mixed sample, wherein the specific reagents (141) are preferably located upstream in the microfluidic device (100). • Denaturing (504) nucleic acids contained in the first mixed sample in a denaturing area (151, 152) of the device (100), in particular in a denaturing chamber, to obtain a denatured sample • Mixing (505) at least a part of the denatured sample with process-specific reagents (142) for the type of nucleic acid amplification, in particular enzymes for nucleic acid amplification, to obtain a second mixed sample, wherein the process-specific reagents (142) are preferably located upstream in the microfluidic device (100). • Performing (506) nucleic acid amplification with at least a part of the second mixed sample in an amplification area, in particular at least one amplification chamber, of the device (100), in particular as isothermal nucleic acid amplification 2. Method (500) according to claim 1, wherein the specific reagents (141) are placed in a first bead chamber (114) and the process-specific reagents (142) are placed in a second bead chamber (124), preferably in dried form, in particular each as part of a bead (141 , 142).

3. Method (500) according to any one of the preceding claims, wherein the denaturing (504) comprises denaturing specific reagents (141), in particular primers, contained in the first mixed sample.

4. Method (500) according to one of the preceding claims, wherein the denaturation (504) is carried out by heating at least a part of the first mixed sample to a temperature higher than 90 °C, preferably higher than 92 °C, most preferably higher than 95 °C, for example 98 °C.

5. Method (500) according to one of the preceding claims, wherein the denaturation (504) takes place in one or more chambers (112, 113) of a first PCR area (101), in particular a first PCR strand, of the microfluidic device (100).

6. Method (500) according to one of the preceding claims, wherein the mixing (505) with the method-specific reagents (142) and / or the carrying out (506) of the nucleic acid amplification takes place in chambers (124, 123) of a second PCR area (102) of the microfluidic device (100).

7. Method (500) according to any of the preceding claims, wherein nucleic acids contained in the sample are purified prior to mixing with the specific reagents to obtain a purified sample.

8. Method (500) according to claim 7, wherein the purification (502) is carried out by separating the nucleic acids from the sample by binding the nucleic acids to a solid phase, in particular to a filter, wherein the nucleic acids are detached from the solid phase, preferably after removal of sample residues with a washing medium, to form a part of the purified sample.

9. Method (500) according to any one of the preceding claims, wherein nucleic acid contained in the sample is released by lysis of cells in the sample.

10. Method (500) according to one of the preceding claims, wherein carrying out (506) the nucleic acid amplification as isothermal amplification comprises, in particular, repeated movement of at least a part of the second mixed sample subjected to amplification between a first amplification chamber (122) and a second amplification chamber (123), in particular to perform an optical selection of the part from the second amplification chamber (123) during the amplification.

11. Microfluidic device (100) for carrying out nucleic acid amplification, in particular according to a method (500) of the preceding claims, comprising reagents (141) specific for nucleic acid amplification, in particular upstream primers, a denaturation area (151, 152), in particular with a denaturation chamber (112, 113), for denaturing nucleic acids contained in the sample mixed with the specific reagents (141) and reagents (141) specific for the type of nucleic acid amplification, in particular comprising enzymes, in particular a polymerase, and at least one amplification chamber (123) for carrying out the nucleic acid amplification.

12. Device (100) according to claim 11, wherein the specific reagents (141) are placed upstream in a first PCR area (101), in particular in a first bead chamber (114), in particular as a detection-specific bead (141), and wherein the process-specific reagents (142) are placed upstream in a second PCR area (102) spaced apart from the first PCR area (101), in particular in a second bead chamber (124), in particular as a process-specific bead (142), 13. Device (100) according to claim 11 or 12, wherein the denaturation area (151, 152), in particular the denaturation chamber (111, 112), and the second PCR area (102), in particular the second bead chamber (124), are arranged in the device (100) relative to each other such that a heating of the denaturation area (151, 152) from a first initial temperature to a denaturation temperature during a denaturation time does not raise the temperature of the second PCR area (102) from a second initial temperature above a Maximum temperature increased.

14. Device (100) according to claim 13; wherein the denaturation temperature is 92 °C, preferably 95 °C, most preferably 99 °C, wherein the denaturation time is 2 minutes, preferably 5 minutes, most preferably 10 minutes, wherein the The maximum temperature is 70 °C, preferably 60 °C, most preferably 50 °C and / or wherein the first and / or the second output temperature is 20 °C, preferably 30 °C, most preferably 40 °C.

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