Method and device for pre-amplifying nucleic acid mixtures

Abstract

The invention relates to a method for pre-amplifying a nucleic acid mixture (30). The method comprises denaturing (21) a nucleic acid mixture (30) to obtain single-stranded nucleic acids (31, 32), hybridizing (22) the single-stranded nucleic acids (31, 32) to chemically modified primers (41, 42), and isothermal pre-amplification (23) of the hybridized nucleic acids (51, 52, 53, 54) using an SD polymerase (61). Furthermore, the invention relates to a microfluidic device comprising an element for receiving a patient sample, an element for pre-amplifying a nucleic acid mixture (20) in the patient sample, and an element for detecting a nucleic acid biomarker in the pre-amplified nucleic acid mixture (30). The device (10) is configured for pre-amplification (23) by the method.

Description

[0001] The present invention relates to a method for pre-amplifying a nucleic acid mixture. Furthermore, the present invention relates to a microfluidic device configured to perform pre-amplification by this method. Prior Art

[0002] In molecular diagnostics, diseases can be detected based on nucleic acids as biomarkers. The nucleic acids can be DNA or RNA. In order to detect as many pathogens as possible simultaneously based on such biomarkers, probe-based methods or sequencing can be used. However, in the case of a small amount of sample input material, these methods must be pre-amplified beforehand, for example, pre-amplification of relevant biomarkers or the total mixture. However, it is not possible to pre-amplify DNA and RNA simultaneously in one reaction.

[0003] Pre-amplification can be carried out by thermophilic enzymes that require periodic temperature changes. This results in an increase in reaction time, so that such pre-amplification reactions are not suitable for use at the point of care. Isothermal amplification reactions can be carried out using mesophilic enzymes. However, their continuous synthesis ability is not as good as that of thermophilic enzymes, so that using them also results in very long reaction times. Summary of the Invention

[0004] A method for pre-amplifying a nucleic acid mixture, especially one contained in a patient sample, includes denaturing the nucleic acid mixture to obtain single strands of nucleic acids. Then, the single strands of nucleic acids are hybridized with chemically modified primers. Isothermal pre-amplification of the thus obtained hybrid nucleic acids is carried out using SD polymerase (strand displacement). SD polymerase is a DNA polymerase suitable for isothermal amplification reactions and is known for its high strand displacement activity in the amplification reaction. This method is based on the discovery that SD polymerase is also suitable for pre-amplification reactions. Using SD polymerase has multiple advantages. It can achieve isothermal pre-amplification, without the low continuous synthesis ability of mesophilic enzymes. In addition, some SD polymerases can tolerate high temperatures during isothermal pre-amplification, which can increase the reaction rate, thus significantly shortening the reaction time. Finally, some SD polymerases can use RNA as a template for amplification in addition to DNA. Therefore, this method requires little or no adjustment for different pathogens or biomarkers to be detected, because DNA and RNA can be generally amplified regardless of the application.

[0005] Before denaturation, the nucleic acid mixture can be separated from a solid phase (such as silica), especially using water or an elution buffer. Denaturation is preferably carried out by heating the nucleic acid mixture dissolved in water, where heating is particularly preferably carried out to a temperature of 75 °C to 98 °C, most preferably to a temperature of 80 °C to 95 °C. Preferably, heating is carried out for a time exceeding 10 seconds, more preferably for a time of 30 seconds to 120 seconds. Thereby, the secondary and tertiary structures of the nucleic acids can be reliably resolved.

[0006] Chemically modified primers for hybridization with single-stranded nucleic acids can preferably be placed together with the nucleic acid mixture before denaturation, particularly preferably by dissolving them in water or elution buffer. They are insensitive to denaturation conditions. Then, hybridization is preferably carried out at a temperature of 4 °C to 50 °C for a period of time, which is preferably more than 10 seconds. Under these conditions, the primers can attach to the single-stranded nucleic acids. The temperature required for hybridization is achieved in particular by cooling the solution of single-stranded nucleic acids obtained by denaturation.

[0007] To prepare isothermal pre-amplification, SD polymerase is added to the hybridized nucleic acid solution. In addition, deoxynucleoside triphosphates (dNTPs) and buffer components can also be specifically added. Deoxynucleoside triphosphates serve as precursor building blocks for nucleic acids in this pre-amplification. The buffer components are used here to set the optimal pH value for pre-amplification. In addition, it is preferred to add single-stranded binding proteins (SSB). Single-stranded binding proteins are proteins that bind to single-stranded DNA to facilitate isothermal pre-amplification by assisting the SD activity of the polymerase.

[0008] Isothermal pre-amplification is preferably started only when the solution of the hybridized nucleic acids and all other added reagents forms a homogeneous reaction mixture. Isothermal pre-amplification is preferably carried out at a constant temperature of 40 °C to 72 °C. The preferred pre-amplification time is 10 minutes to 120 minutes. The higher the temperature during pre-amplification, the faster it proceeds, and generally a shorter pre-amplification time can be advantageously selected. Compared with pre-amplification using a mesophilic enzyme at 40 °C or lower, the reaction time can be significantly shortened. In particular, at a temperature of 45 °C to 65 °C, the reaction is significantly accelerated. Another advantage of this method is that it can be used for pre-amplification of RNA even at such a high temperature, while other methods only achieve pre-amplification of RNA at lower temperatures.

[0009] The primers are preferably primers having at least one modification selected from LNA (locked nucleic acid), MGB (minor groove binder), C-5 propynyl deoxycytidine, C-5 propynyl deoxyuridine, aminoethylphenoxazine deoxycytidine, 5-methyl deoxycytidine, 2-amino deoxyadenosine, trimethoxystilbene, pyrene, and spermine. Particularly preferred are ZNA primers (Zip Nucleic Acids). These are spermine-modified primers. Their advantage is that they form a particularly temperature-stable hybridized nucleic acid with single-stranded nucleic acids and can use a higher hybridization temperature without sequence extension of the primers. In addition, at the corresponding spermine loading of the primers, they reduce the self-hybridization of the primers. This is particularly advantageous when the primers used are short and isothermal pre-amplification is carried out at a temperature of at least 45 °C.

[0010] In a preferred embodiment of the method, the primers are modified primers with a random sequence order (random primers). These, in particular, have a defined chain length (preferably 6 to 15 bases) and the advantage of primers consisting of a purely random sequence order is that they can cover all regions of the nucleic acid template to be amplified with a high probability. They enable isothermal pre-amplification to be carried out as uMDA (universal multiple displacement amplification).

[0011] In another preferred embodiment of the method, in addition to using modified primers with a random sequence, primers with a specific sequence are also used to hybridize with single-stranded nucleic acids. By additionally using the specific primers, ssMDA (semi-specific multiple displacement amplification) is achieved. These primers with a specific sequence can also carry modifications that increase the processive synthesis ability of SD polymerase. In particular, these are spermine groups. Particularly preferred are ZNA primers with a specific sequence, which increase the processive synthesis ability of SD polymerase and repel each other when correspondingly loaded. This modification helps to prevent unwanted interactions between primers with a specific sequence. In addition, a large number of different primers with a specific sequence can thus be used.

[0012] Primers with a specific sequence are understood to be primers that have not only a defined chain length but also a defined sequence. In particular, the sequence can be selected such that it binds to a specific position on the nucleic acid, for example flanking one or more targets, thus facilitating the enrichment of one or any number of targets relative to the background. Alternatively or additionally, the sequence can be selected such that it binds to a nucleic acid sequence to which SD polymerase has a lower processive synthesis ability compared to other nucleic acid sequences, for example due to difficult sequences with a high GC content. By enhancing the pre-amplification of sequences that were originally amplified only insufficiently, not only universal pre-amplification but also more uniform pre-amplification can be achieved.

[0013] Primers with a specific sequence preferably have at least one selection marker, such as biotin. Such a selection marker is advantageous when the primer is specific for the target and not only used to improve consistency. In this case, its selection marker can be used to purify or enrich the amplification product of the specific primer.

[0014] For both modified primers with a random sequence and primers with a specific sequence, it is preferred that the primers have a single-strand break cleavage site. After generating a single-strand break by an appropriately added endonuclease (nicking enzyme), the single-strand break cleavage site provides an additional amplification start point for SD polymerase. This enables pre-amplification to continue at these sites without further primer hybridization, which increases the processive synthesis ability.

[0015] For pre-amplification, an endonuclease is added to the SD polymerase for this purpose. The endonuclease is preferably selected from Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nt.BsmAI, Nt.BspQI, Nb.BsrDI, Nb.BssSI, Nt.BstNBI, Nb.BtsI and Nt.CviPII.

[0016] For example, the SD polymerase can be an enzymatic mutant of the DNA polymerase of Bacillus subtilis phage Phi2, in particular EquiPhi29, or an enzymatic mutant of the DNA polymerase of Pyrococcus furiosus, in particular Vent(exo-). The advantage of the SD polymerase Vent(exo-) is that it remains stable at temperatures up to 100 °C and has processive synthesis ability. Therefore, it can be added to the nucleic acid mixture before denaturation without being damaged by the high denaturation temperature. However, these SD polymerases do not have RNA processive synthesis ability.

[0017] Preferably, the nucleic acid mixture is a mixture containing both DNA and RNA so as to enable the general pre-amplification reaction possibilities provided by the present method. The SD polymerase is preferably an enzymatic mutant of the DNA polymerase of Bacillus stearothermophilus. In addition to their processive synthesis ability for DNA, these SD polymerases also exhibit processive synthesis ability for RNA. Particularly preferred enzymatic mutants of the DNA polymerase of Bacillus stearothermophilus are selected from Bst, Bst2.0 and Bst 3.0. Among these enzymatic mutants, Bst 3.0 has the highest RNA processive synthesis ability, and thus Bst 3.0 is most preferred.

[0018] If an SD polymerase with 3'→5' exonuclease activity is used, the primer can be modified and thus protected with a 3'-terminal thiophosphate group (PTO).

[0019] Although the SD polymerase is capable of pre-amplifying RNA in addition to pre-amplifying DNA, it is preferred to also use at least one reverse transcriptase during isothermal pre-amplification. In particular, these are RTx or SSIV. However, in principle, all reverse transcriptases with processive synthesis ability under reaction conditions similar to those of the SD polymerase used are conceivable. Thereby, the pre-amplification of RNA is assisted in a targeted manner so that the required pre-amplification time can be further shortened. If an SD polymerase without RNA processive synthesis ability is used, a reverse transcriptase also needs to be used to have processive synthesis ability for RNA in addition to DNA.

[0020] Since this method can perform isothermal preamplification in only a single reaction chamber where denaturation, hybridization, and isothermal preamplification occur, it can be easily integrated into a microfluidic device or a lab-on-a-chip system, enabling automated analysis at the point of care without additional equipment. For this purpose, a microfluidic device is provided, which has an element for receiving a patient sample. Another element can be optionally used to purify the nucleic acid mixture in the patient sample and make it available in a pure form for preamplification. Another element is provided for preamplifying the nucleic acid mixture in the patient sample to perform preamplification using this method. Preferably, reagents for preamplification can be stored in this element, especially in a dried state in the form of beads. Here, preamplification can be performed directly using the patient sample without a prior purification element, or it can also be performed using a purified nucleic acid mixture. Finally, an element is provided for detecting nucleic acid biomarkers in the preamplified nucleic acid mixture. For example, target detection can be performed here via isothermal amplification by adding specific primers and probes. However, the preamplified nucleic acid can also be diluted and dispensed into separate cavities to enable the simultaneous and individual detection of multiple targets. Detection methods using qPCR, CRISPR / Cas, NGS, or microarrays can also be used for target detection. Brief Description of the Drawings

[0022] Embodiments of the present invention are shown in the drawings and are explained in more detail in the following description.

[0023] Figure 1 A microfluidic device according to an embodiment of the present invention is schematically shown.

[0024] Figure 2 A flow of a method according to an embodiment of the present invention is schematically shown.

[0025] Figure 3 A flow of a method according to another embodiment of the present invention is schematically shown.

[0026] Figure 4 A flow of a method according to yet another embodiment of the present invention is schematically shown.

[0027] Figure 5 A flow of a method according to yet another embodiment of the present invention is schematically shown.

[0028] Figure 6 A microfluidic device according to another embodiment of the present invention is schematically shown.

[0029] Figure 7 A flow of a method according to yet another embodiment of the present invention is schematically shown.

[0030] Figure 8 A flow of a method according to yet another embodiment of the present invention is schematically shown.

[0031] Embodiments of the present invention

[0032] In one embodiment of the present invention, a microfluidic device is provided, which is designed, for example, as a disposable cartridge for an analysis device. Such a cartridge has a fluid layer, a pneumatic layer, and an elastomeric membrane disposed between the fluid layer and the pneumatic layer. The fluid layer and the pneumatic layer each have a substrate composed of, for example, polycarbonate. For example, the elastomeric membrane is composed of thermoplastic polyurethane. The fluid layer contains channels and chambers that are arranged to transport reaction liquids and reaction mixtures in molecular diagnostics. In addition, chemicals for performing chemical reactions are stored upstream of the fluid layer. The pneumatic layer has channels that open at the elastomeric membrane and have openings on the outside of the pneumatic layer. Through these openings, the channels can be connected to the pneumatic manifold of the analysis device. When an overpressure is generated in one of the individually controllable channels of the pneumatic layer using the pneumatic manifold, the elastomeric membrane deflects into the fluid layer. Conversely, if a negative pressure is generated in the channel, the elastomeric membrane deflects into the pneumatic layer. In this way, the fluid flow in the fluid layer can be manipulated.

[0033] Figure 1 The figure in shows the elements of the fluid layer of the microfluidic device 10. The first element 11 is configured to receive a patient sample. For this purpose, it has an opening on the outside of the fluid layer through which the patient sample can be introduced and absorbed on silica gel in the first element 11. The first element 11 is fluidly connected to a second element 12, which is used to purify the patient sample. The second element 12 is fluidly connected to a third element 13, which is used to pre-amplify the nucleic acid mixture contained in the patient sample. The third element 13 is fluidly connected to a fourth element 14, which is used to purify the pre-amplified product. The fourth element 14 is fluidly connected to a fifth element 15, which is used to detect nucleic acid biomarkers in the purified pre-amplified nucleic acid mixture.

[0034] Figure 2Shows the method steps 21 - 24 carried out in the microfluidic device 10 in a first embodiment of the method according to the present invention. The nucleic acid mixture 30 contains DNA 31 and RNA 32 as nucleic acids, which have secondary and tertiary structures. The nucleic acid mixture 30 is eluted in the first element 11 with an aqueous solution of ZNA primers and pumped into the second element 12. There, the first method step 21 involves denaturing the nucleic acid mixture 30 by heating the solution to 95 °C for one minute. Single-stranded DNA 31 and single-stranded RNA 32 are thereby obtained, which are present in the solution together with the ZNA primers 41. For example, the ZNA primer 41 is a modified primer (N9-ZNA3) with three spermine groups on an N9 oligonucleotide (random sequence order of 9 bases). The next method step 22 involves the hybridization of the single-stranded DNA 31 and single-stranded RNA 32 with the ZNA primers 41. For this purpose, the solution is cooled to a temperature of, for example, 40 °C and held at this temperature for one minute. Primer-hybridized DNA 51 and primer-hybridized RNA 52 are thereby obtained. In the next method step 23, the hybridized DNA 51 and hybridized RNA 52 are pre-amplified. For this purpose, first a further reaction solution is supplied from a chamber not shown in the figure into the solution and uniformly mixed with the solution of the hybridized DNA 51 and hybridized RNA 52. The further reaction solution contains, for example, an SD polymerase 61 called Bst 3.0, as well as dNTP, SSB (single-stranded binding protein) and buffer. Then the homogeneous reaction mixture is heated to a temperature of 50 °C for, for example, 30 minutes. Here, the SD polymerase 61 pre-amplifies the sequences hybridized using the ZNA primers 41 by strand displacement of both the hybridized DNA 51 and hybridized RNA 52. Next, a detection reaction 24 is carried out. For this purpose, the reaction mixture is pumped into the third element 13. There, for example, it is distributed in a plurality of cavities of a microarray, and a qPCR reaction is carried out there as a detection method. Here, the third element 13 has a window on its side facing away from the elastomeric membrane, which window faces the sensor of the analysis device. This enables the reaction carried out in the cavities of the microarray to be evaluated by fluorescence spectroscopy. Figure 1 A chamber not shown in the figure is supplied into the solution and uniformly mixed with the solution of the hybridized DNA 51 and hybridized RNA 52. The further reaction solution contains, for example, an SD polymerase 61 called Bst 3.0, as well as dNTP, SSB (single-stranded binding protein) and buffer. Then the homogeneous reaction mixture is heated to a temperature of 50 °C for, for example, 30 minutes. Here, the SD polymerase 61 pre-amplifies the sequences hybridized using the ZNA primers 41 by strand displacement of both the hybridized DNA 51 and hybridized RNA 52. Next, a detection reaction 24 is carried out. For this purpose, the reaction mixture is pumped into the third element 13. There, for example, it is distributed in a plurality of cavities of a microarray, and a qPCR reaction is carried out there as a detection method. Here, the third element 13 has a window on its side facing away from the elastomeric membrane, which window faces the sensor of the analysis device. This enables the reaction carried out in the cavities of the microarray to be evaluated by fluorescence spectroscopy.

[0035] In the second embodiment of the method of the present invention, it is provided that the reaction solution mixed with the mixture of hybridized DNA 51 and hybridized RNA 52 in the second element 12 contains reverse transcriptase 62 in addition to the components provided in the first embodiment. It is, for example, RTx. In the isothermal pre-amplification 23 step, as in the first embodiment, the hybridized DNA 51 is pre-amplified by SD polymerase 61. Although the SD polymerase 61 also pre-amplifies each hybridized RNA strand 52 as in the first embodiment, in the second embodiment, the pre-amplification of the hybridized RNA strand 52 is carried out after reverse transcription into cDNA by the reverse transcriptase 62. The further method steps of the second embodiment of the method are consistent with those of the first embodiment.

[0036] Although the pre-amplification 23 is carried out by uMDA in the first two embodiments, the method according to the present invention also realizes ssMDA in a further embodiment:

[0037] In Figure 4In the third embodiment of the method of the present invention as shown, the elution solution introduced into the first element 11 contains, in addition to the modified ZNA primer 41 having a random sequence order, a modified primer 42 having a specific sequence. The modified primer 42 having a specific sequence herein has a sequence capable of hybridizing the sequences of DNA 31 and RNA 32 (while the SD polymerase 61 has only a low processive synthesis ability for them). Although the SD polymerase can amplify most of the sequences of DNA 31 and RNA 32 a thousand-fold, for example, by using the modified ZNA primer 41 having a random sequence, these nucleic acids may also have sequences that are amplified only twenty-fold. At this time, it is provided that the modified ZNA primer 42 having a specific sequence is a primer having a sequence specific for these DNA sequences and RNA sequences that can be amplified only weakly. At the hybridization 22, on the one hand, the hybridized DNA strand 51 is formed as in the first two embodiments, which carries only the modified ZNA primer 41 having a random sequence, and the hybridized RNA strand 52 is formed, which carries only the ZNA primer 41 having a random sequence. In addition, a hybridized DNA strand 53 is also formed, which carries the modified ZNA primer 41 having a random sequence and the modified primer 42 having a specific sequence. In addition, a hybridized RNA strand 54 is formed, which carries the modified ZNA primer 41 having a random sequence and the modified primer 42 having a specific sequence. At the pre-amplification 23, the SD polymerase 61 forms the same pre-amplified product 71 as can also be obtained in the first two embodiments of the method according to the present invention from the hybridized DNA strands 51 and RNA strands 52 (which carry only the ZNA primer 41 having a random sequence). In addition, the SD polymerase 61 forms a second amplified product 72 at the sequence where the modified primer 42 having a specific sequence is attached by pre-amplifying the hybridized DNA strands 53 and RNA strands 54 (which carry the modified ZNA primer 41 having a random sequence and the modified primer 42 having a specific sequence), which has an increased proportion of sequences that are difficult to amplify. In other respects, the process of the method according to the third embodiment of the present invention corresponds to the uMDA according to the first embodiment of the present invention.

[0038] In Figure 5 In the fourth embodiment of the present invention as shown, the fourth embodiment is modified such that, as in the second embodiment of the method, the reaction solution mixed with the hybridized nucleic acids 51 - 54 in the second element 12 further contains a reverse transcriptase 62. At the pre-amplification 23, the hybridized DNA strands 51, 53 are only pre-amplified by the SD polymerase 61 at this time, while the hybridized RNA strands 52, 54, although also partially pre-amplified by the SD polymerase 61, are mainly pre-amplified after being reverse transcribed into cDNA by the reverse transcriptase 62. In other respects, the process of the fifth embodiment of the method according to the present invention corresponds to the process of the third embodiment.

[0039] Figure 6 An element of the fluid layer showing another embodiment of the microfluidic device 10. Different from the Figure 1 device shown, a fifth element 15 for purifying the pre-amplified product is arranged between the third element 13 and the fourth element 14. The fifth element 15 is arranged for streptavidin-based purification to give a particularly favorable ratio of relevant biomarker sites compared to less relevant sites. This can increase the sensitivity and specificity in the downstream detection method in the third element 13.

[0040] When using the device, the third and fourth embodiments of the method are modified to fifth and sixth embodiments. The modified primers 42 with specific sequences have sequences selected such that they bind to biomarker-related sites of, for example, viruses, bacteria, fungi, and tumors or sequences adjacent to these sites, and the microfluidic device 10 is arranged for detecting these sites. These modified primers 42 with specific sequences have, for example, biotin at the 5'-end as a selection marker. In these modified embodiments, the second amplification product 72 is specific for these sequences. After the pre-amplification 23 is completed and before the next detection step 24, streptavidin-based intermediate purification is arranged in the fifth element 15 to detect the relevant biomarker with high sensitivity and specificity in the detection step 24.

[0041] Figure 7 and Figure 8 shows a further modification of the third and fourth embodiments of the present invention to seventh and eighth embodiments. Here, the modified primers 42 with specific sequences each have, for example, the 5'...GGATCNNNNN...3' sequence as a single-strand break cleavage site. In the third element 13, Nt.AlwI is added as the endonuclease 13. After the second pre-amplified product 72 exists in double-stranded form 73 after multiple rounds of pre-amplification, the endonuclease 63 binds to the single-strand break cleavage site and introduces a single-strand break. The SD polymerase 62 recognizes this and uses it as an attack point for further pre-amplification. If the single strand obtained thereby becomes double-stranded again, the endonuclease 63 will recognize the single-strand break cleavage site on the same molecule again and repeat the process. Therefore, the yield of the second pre-amplified product 72 can be further increased compared to the third and fourth embodiments.

[0042] In all embodiments of the method according to the present invention, the result of the detection reaction occurring in the fourth element 14 is finally output via the user interface on the analysis device. The microfluidic device 10 can be discarded as a disposable article after its use.

Claims

1. A method for pre-amplifying a nucleic acid mixture (30), which comprises the following steps: - denaturing (21) the nucleic acid mixture (30) to obtain single-stranded nucleic acids (31, 32), - hybridizing (22) the single-stranded nucleic acids (31, 32) with chemically modified primers (41), and - performing isothermal pre-amplification (23) of the hybridized nucleic acids (51-54) using SD polymerase (61).

2. The method according to claim 1, wherein, the isothermal pre-amplification (23) is carried out at a temperature of 40°C to 72°C.

3. The method according to claim 2, wherein, the isothermal pre-amplification (23) is carried out at a temperature of 45°C to 65°C.

4. The method according to any one of claims 1 to 3, wherein, the primer (41) is a ZNA primer.

5. The method according to any one of claims 1 to 4, wherein, the primer (41) is a modified primer with a random sequence.

6. The method according to any one of claims 1 to 5, wherein, the single-stranded nucleic acids (31, 32) are also hybridized (22) with primers (42) having specific sequences.

7. The method according to claim 6, wherein, each of the primers (42) having specific sequences (42) has at least one selection marker.

8. The method according to any one of claims 1 to 7, wherein, the primers (41, 42) have single-strand cleavage sites for endonucleases (63).

9. The method according to any one of claims 1 to 8, wherein, the nucleic acid mixture (30) contains DNA (31) and RNA (32).

10. The method according to claim 9, wherein, the SD polymerase (61) is an enzyme mutant of the DNA polymerase of Bacillus stearothermophilus.

11. The method according to claim 10, wherein, the SD polymerase (61) is Bst 3.

0.

12. The method according to any one of claims 9 to 11, wherein, at least one reverse transcriptase (62) is additionally used in the isothermal pre-amplification (23).

13. A microfluidic device (10), which comprises an element (11) for receiving a patient sample, an element (13) for pre-amplifying a nucleic acid mixture (30) in the patient sample, and an element (14) for detecting nucleic acid biomarkers in the pre-amplified nucleic acid mixture (30), wherein, the device (10) is configured to perform pre-amplification (23) by the method according to any one of claims 1 to 12.

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