Reaction mixtures, methods and kits for performing quantitative real-time PCR

Abstract

In the case of providing a reaction mixture for a reaction batch for quantitative real-time PCR, the reaction mixture contains at least one target DNA (11) corresponding at least in part to a DNA fragment to be quantified, and at least one reference DNA (12) having a specific sequence and a specific amount, and at least two different fluorescent probes having different sequences, which generate signals at different wavelengths, and primers and deoxynucleotides and DNA polymerase. The target DNA (11) and the reference DNA (12) have the same primer binding sites (13, 14) and different probe binding sites (17, 18). At least one of the fluorescent probes is configured to bind to a target DNA (11) fragment outside the primer binding sites (13, 14) in the amplicon, and at least one of the fluorescent probes is configured to bind to a reference DNA (12) fragment outside the primer binding sites (13, 14) in the amplicon.

Description

[0001] The present invention relates to a reaction mixture providing a reaction batch for performing quantitative real-time PCR, as well as to a method for performing quantitative real-time PCR and to a kit. Prior art

[0002] The polymerase chain reaction (PCR) is a very sensitive bioanalytical method. With the help of an enzyme, namely a DNA polymerase, DNA is amplified based on a DNA sequence as a template. Here, the product formed in each cycle is used as a template for the next cycle. The DNA to be replicated is referred to as a template (template DNA) here. In addition, so-called primers are required, which respectively specify the starting point of DNA synthesis on a single strand of DNA. DNA synthesis is catalyzed by a temperature-stable DNA polymerase using deoxynucleotides. For each PCR cycle, double-stranded DNA is first denatured (melted), and then primer hybridization is performed, that is, the primer can bind to the complementary sequence fragment of the single-stranded DNA (primer annealing). Next, the DNA polymerase is attached, and complementary extension of the primer occurs in the so-called extension step (extension). These individual steps are controlled by temperature cycles.

[0003] One embodiment of PCR is real-time PCR (qPCR), in which the course of the reaction can be followed with the aid of fluorescent probes. Real-time PCR allows the quantification of the starting amount of template DNA. Usually, for this purpose, a reference measurement is required that is carried out in parallel reaction batches. In addition to the quantitative reference measurement, qualitative controls are also carried out in a standard manner in order to be able to exclude false positive or false negative results.

[0004] Invention Disclosure

[0005] Advantages of the invention

[0006] The present invention provides a reaction mixture, which is configured to provide a reaction batch for performing quantitative real-time PCR. Using this reaction mixture, a DNA sequence, such as a DNA sequence of a gene fragment, can be quantitatively measured, wherein a parallel standard reaction has been integrated into each reaction batch or reaction mixture, and therefore there is no need to carry out a parallel standard reaction and a control reaction. Here, a special advantage is that the quantitative and quality control can be measured in the same reaction batch (PCR batch). In this regard, a reaction batch should be understood to mean that the reaction is carried out in a reaction vessel. Therefore, there is no need to carry out a reference measurement, thereby greatly saving the cost of the PCR batch. This can provide considerable advantages, for example, particularly in point-of-care (PoC) applications, because in such applications, it is usually directly examined for the patient. In conventional methods, it is usually necessary to prepare corresponding reference measurements for each patient and to carry out in parallel. When using the reaction mixture of the present invention, this is not necessary. Therefore, the reaction mixture of the present invention and the method that can be carried out therewith can be advantageously used, particularly in medical diagnosis.

[0007] The reaction mixture of the present invention comprises at least one target DNA, which corresponds at least in part to the DNA sequence to be quantified. The target DNA is also referred to as a quantifier hereinafter. In addition, the reaction mixture comprises at least one reference DNA, which has a specific artificial sequence and is present in the reaction mixture in a specific amount. The reference DNA is also referred to as an artificial quantifier hereinafter. In addition, at least two different fluorescent probes with different sequences are provided, which generate signals at different wavelengths. In addition, primers, deoxynucleotides and thermostable DNA polymerases are included. Depending on the application, the primer is one or more primer pairs. The target DNA and the reference DNA have the same primer binding site (primer hybridization site). In addition, different probe binding sites are provided on the target DNA and the reference DNA, wherein the probe binding site is located outside the primer binding site in each amplicon. Here, the term amplicon generally refers to the DNA to be replicated. At least one of the fluorescent probes is provided for hybridizing or binding with a target DNA fragment outside the primer binding site in the amplicon. At least one of the fluorescent probes is provided for hybridizing or binding with a reference DNA fragment outside the primer binding site in the amplicon. Therefore, one of the fluorescent probes binds to the target DNA, while the other fluorescent probe binds to the reference DNA. The fluorescent probe is preferably a single-stranded DNA sequence fragment, each of which is coupled to at least one reporter dye molecule and at least one quencher molecule. The working principle of this fluorescent probe known per se is based on: the fluorescent signal is extinguished due to the spatial proximity of the reporter dye molecule and the quencher molecule when the fluorescent probe is intact or the DNA molecule of the fluorescent probe is intact. During the PCR reaction, the fluorescent probe is attached to the respective complementary fragments of the template DNA (outside the primer binding site). During the amplification process, the DNA polymerase moves along the strand to be replicated of the template DNA, and inevitably encounters the accumulated fluorescent probe here. Through the 5'-3' exonuclease activity of the DNA polymerase, the fluorescent probe is cut off, thereby eliminating the spatial proximity of the reporter dye molecule and the quencher dye molecule, thereby generating a fluorescent signal. Therefore, the completion of amplification can be inferred from this measurable fluorescent signal. Therefore, by using two different fluorescent probes, one of which interacts with the target DNA or the other interacts with the reference DNA, both the target DNA-based amplification and the reference DNA-based amplification can be tracked by different fluorescent signals in the reaction batch. In this case, it is expedient to select the fluorophores of the probes in such a way that the colors or fluorescence signals can be distinguished from one another by means of a detector arrangement and suitable filter sets.

[0008] Suitably, the specific artificial sequence of the reference DNA is not identical (orthogonal) to the sequence of the target DNA, i.e. not homologous, wherein the GC content, i.e. the total content of guanine (G) and cytosine (C) in the sequence is preferably as identical as possible to the GC content of the target DNA, independently of its position in the sequence itself. "As identical as possible" is understood in this context to mean that the deviation in percentage of the GC content of the target DNA and the reference DNA may be, for example, at most 15%, preferably at most 10%. It is further preferred that the base pair length of the target DNA sequence and the reference DNA sequence is as identical as possible, wherein, for example, a maximum deviation of at most 15%, preferably at most 10%, may be acceptable.

[0009] The concept on which the reaction mixture for quantitative real-time PCR is based is that an artificial reference DNA of a specific composition and amount is added to the reaction batch. The reference DNA allows internal calibration and can also perform the tasks of positive and negative controls. In principle, multi-template PCR is performed, wherein a variety of different specific amplicons are produced in parallel in the amplification reaction. At least two templates are provided, namely target DNA and reference DNA. In principle, the amplification of different templates is performed with only one primer pair that hybridizes with the target DNA and the reference DNA. By using different probes, wherein the probe for the target DNA and the probe for the reference DNA are specific (or optionally multiple probes), different fluorescent signals or fluorescent colors can be detected, wherein the test results can be obtained by the ratio of the different fluorescent signals to each other.

[0010] Due to the integrated reference and control, the PCR process carried out with the reaction mixture is particularly suitable for automation and miniaturization, particularly in the scope of microfluidic applications. It may be particularly advantageous that the different components of the reaction mixture are provided in a lyophilized form. Therefore, it is particularly possible to provide and present target DNA and / or reference DNA and / or primers and / or deoxynucleotides and / or DNA polymerase in a lyophilized form. This can be achieved, for example, in the form of one or more so-called lyophilized beads. Lyophilized beads can generally be understood to refer to lyophilized materials that are pressed into a spherical shape after manufacturing (after the manufacturing, the material is usually present as a powder). Therefore, components required for PCR batches, particularly DNA polymerase, deoxynucleotides, target DNA and reference DNA and reaction buffer components and optional primers and / or probes can be provided, for example, in a lyophilized form. In this way, the PCR process can be directly started in a very user-friendly manner by adding samples to be quantified and optional other required components. In particular, the provision of lyophilized forms is very conducive to automated applications.

[0011] Providing the reaction mixture or at least some parts of the reaction mixture in the form of lyophilized beads also has the advantage that the manufacturing costs of the lyophilized beads as well as the development costs can be greatly reduced by integrating the standards and / or controls into the reaction batch. Due to the reduction in the amount of the required reaction batch, integration into a microfluidic system is also particularly advantageous, since fewer reaction chambers are required compared to conventional PCR processes and no additional chambers have to be expanded for the microfluidic platform. In addition, the run time of the real-time PCR can be shortened, since the concept on which the invention is based makes it possible to introduce the reaction conditions into a particularly effective or ideal reaction range by means of a predetermined amount of template, so that a fluorescent signal can always be expected.

[0012] Another particular advantage of the PCR process described here is that, due to the integration of the standards and / or controls into the reaction batch, the conditions for the standards, controls and actual samples are identical to the DNA to be quantified. If, for example, air bubbles are present in the reaction batch (which can be the case in rare cases, for example, in microfluidic systems), the associated effects on the reaction efficiency are identical for all templates, i.e., for example, for quality control, calibration and for the actual sample reaction, so that the entire experiment is comparable and evaluable in each case.

[0013] In a traditional way, a standard line is usually created in the scope of quantitative real-time PCR, wherein at least three different concentrations are usually assumed for this standard line, and these concentrations are within the expected sample concentration range. For statistical reasons, more concentrations are usually selected for standard reactions, and these standard reactions are also processed in a manner of multiple executions. In the concept for real-time PCR described herein, calibration is performed by means of the multi-signal concept of different fluorescent probes and their ratios to each other, so only one reaction is required and quantification can still be performed. This has considerable advantages in terms of the operating costs and process costs required for this and in terms of the favorable minimization of expensive chemicals and sample requirements or the required small sample amount.

[0014] In a preferred embodiment of the reaction mixture, the amount of the reference DNA can be present at a concentration corresponding to the detection limit of the DNA fragment to be quantified. In addition, depending on the application, the target DNA and the reference DNA can be provided to be present in a 1:1 ratio and in addition in a specific amount.

[0015] The present invention also includes a method for performing quantitative real-time PCR, wherein at least one reaction mixture as described above is used in the method. Typically, an actual sample of nucleic acid material (optionally) comprising a DNA fragment to be quantified is also added to the reaction mixture. The PCR process is carried out as a double reaction to a certain extent with this complete reaction batch, wherein the PCR cycle is carried out in a manner known per se by the change of temperature in a thermal cycle process known per se. Here, on the one hand, the target DNA and the DNA fragment to be quantified (as long as it is present in the sample) and the reference DNA are amplified. By detecting and evaluating the fluorescent signals of different fluorescent probes, wherein these signals are specific to the target DNA (and the DNA fragment to be quantified at the same time) and the reference DNA, the target DNA and the actual sample of the DNA fragment to be quantified at the same time and the amplification of the reference DNA can be detected and can be distinguishably tracked. Test results, especially quantitative test results, can be determined from the ratio between these signals.

[0016] In a particularly preferred embodiment of the method, the method is carried out in a PCR array with a plurality of array containers. This can be particularly advantageously carried out in microfluidic applications, which can also be used for automation in particular. Here, different reaction mixtures can be loaded into each array container of the PCR array, so that the maximum multiple degree can be achieved. The loading can be realized, for example, by dipping different reaction mixtures into each array container. Thus, in particular in the microfluidic PCR array, a sample solution with a DNA fragment to be quantified or a nucleic acid material to be tested can be added to the array as a whole. In this way, the sample solution arrives at each individual array container and forms a respective reaction batch with each different reaction mixture. The special advantage here is that each reaction chamber does not have to be filled and controlled separately. The problem existing in the conventional method is that, in the array container that is provided for standard reaction in the PCR array, sample material is not allowed to be housed. In this respect, what is usually needed in the conventional PCR array is that each reaction chamber must be filled and controlled separately, wherein the filling of the reaction container for standard reaction is different from the reaction chamber that is provided for the PCR process with the actual sample. In contrast, a PCR array for carrying out a PCR process according to the concept described herein allows, on the one hand, significantly larger numbers of PCR batches with the samples to be measured in one array, since no separate reaction batches need to be provided for the standard reactions. On the other hand, as described above, the concept of the present application also allows the entire array as a whole to be filled with sample solution.

[0017] Another particular advantage of the concept for real-time PCR described herein is that the reaction system does not have to be calibrated for the individual light sources, since the test result is derived from the ratio of the signals, which is attributed to the ratio maintained during the individual amplification processes. In the case of different device types, the light sources and optical detectors are usually different. Therefore, in the traditional way, calibration measurements are required for each device type. Even in devices in which two identical LED light sources are installed, these two light sources must be calibrated in the traditional way so that they provide the same absolute numbers required for evaluation via a standard line. In the concept of real-time PCR according to the invention, these complex calibration measurements are omitted because the operation is performed using relative ratios within a batch.

[0018] In medical applications, particularly in medical diagnosis, the sample amount obtained from the patient is usually small. In addition, the analytical system used in instant detection applications is provided for small space requirements and should have a high degree of automation as much as possible to reduce operating costs. In this regard, the microfluidic implementation of the PCR batch described herein is particularly suitable for these applications, wherein, it can be automated, miniaturized and parallelized, which reduces the cost in the application on the one hand, and also minimizes the possibility of error during erroneous operation. In addition, a small sample amount can be transferred to a small volume to make the reaction concentration larger. In the conventional method, parallelization is associated with the challenge during operation, because the distribution of the reaction mixture and the pre-storage and preparation of the required chemicals are usually difficult due to miniaturization. The real-time PCR according to the concept described herein minimizes the cost in the PCR process operation, because the number of the reaction batch is reduced to basically one batch on the one hand. Here, the required reagent can be easily pre-stored in, for example, a chip lab system. Due to freeze drying, reagents can also be provided, for example, in the form of freeze-dried beads at room temperature and in the smallest space.

[0019] In a particularly preferred embodiment of the method, the method can be used for a nested PCR process. The nested PCR process comprises pre-amplification and at least one subsequent detection reaction in a manner known per se. At this, the concept described herein can be used to estimate the amount of PCR products pre-amplified when using a target DNA and a reference DNA in a batch. At this, the target DNA and the reference DNA can be designed in such a way that the first primer pair is used for pre-amplification, and at least one second primer pair is used for (one or more) detection reactions. The target DNA and the reference DNA each have a sequence fragment (primer binding site) complementary to the primer sequence, wherein the complementary sequence fragment of the second primer pair (primer binding site for the primer pair of the (one or more) detection reactions) is located within the complementary sequence fragment of the first primer pair (primer binding site for the primer pair of pre-amplification). This means that the primer binding sites for various primer pairs are nested in the target DNA and the reference DNA to a certain extent.

[0020] The nested PCR process can be used in particular for point mutation detection. Here, after pre-amplification, the amount of the PCR product thereof is determined according to the concepts described herein, a detection reaction can be performed using mutation-sensitive primers and / or mutation-sensitive fluorescent probes.

[0021] The nested PCR process can be a multiplex process, in which at least two specific gene fragments in the genome should be detected. Here, for the quantitative preamplification, a control reaction can be performed, in which a control exon is amplified from the genome in parallel. In this case, the target DNA and the reference DNA are matched with the control exon, wherein the amount of amplification of the gene fragment to be detected in the preamplification process is inferred from the quantitative amplification of the control exon according to the concept described herein.

[0022] In general, the concept for the PCR process described herein can integrate not only quantitative standard lines and quality reactions, but also reference measurements and threshold measurements, which are necessary, for example, in the determination of point mutations in oncology. Although various DNA fragments to be tested require two color channels in the detection, the concept allows multiple modes and can solve a variety of different DNA fragments (targets) in one batch. In particular, in the scope of nested PCR with pre-amplification and subsequent qualitative measurements such as point mutation analysis, the method can be applied in such a way that the starting amount of the second reaction is estimated by quantitatively measuring the pre-amplification. Here, the two PCR processes, i.e., pre-amplification and subsequent detection reactions, can be fully automatically connected to each other in a microfluidic system without the need to measure the DNA concentration separately in an intermediate step, or without the need to purify the PCR products produced during pre-amplification. The nested PCR process can be designed, for example, in such a way that the optimal DNA concentration for the subsequent (one or more) detection reactions is set by dilution according to the estimation of the amount of PCR products from the pre-amplification. This can be performed, for example, in situ, also in an automated manner.

[0023] Finally, the present invention includes a kit for performing quantitative real-time PCR. The kit contains at least one target DNA, which corresponds at least in part to the DNA sequence to be quantified. In addition, the kit contains at least one reference DNA, which has a specific artificial sequence and a specific amount. In addition, there are at least two different fluorescent probes with different sequences, which generate signals at different wavelengths. Optionally, primers and / or deoxynucleotides and / or DNA polymerases and / or buffer components can be set. The target DNA and the reference DNA have the same primer binding site, but have different probe binding sites, wherein the probe binding sites are located outside the primer binding sites in the respective amplicons. At least one of the fluorescent probes is configured to hybridize (bind) with the target DNA fragment outside the primer binding site in the amplicon, and at least one of the fluorescent probes is configured to hybridize (bind) with the reference DNA fragment outside the primer binding site in the amplicon. The components of the kit can be provided in particular in a lyophilized form, for example in the form of lyophilized beads. For other features of the kit, refer to the above description.

[0024] Other features and advantages of the present invention are apparent from the following description of exemplary embodiments. In this case, the individual features can be realized individually or in combination with one another.

[0025] In the attached picture:

[0026] Figure 1 A schematic diagram of the design of target DNA and reference DNA is shown to illustrate the basic principle of the concept of performing quantitative real-time PCR;

[0027] Figure 2A schematic diagram showing template DNA for quantitative real-time PCR and possible test results when quantifying a specific DNA fragment in a sample;

[0028] Figure 3 shows a schematic diagram of the DNA template used for quantitative real-time PCR ( Figure 3A ), and a schematic diagram of possible test results when applying the concept in the context of quantitative nested PCR ( Figure 3B );

[0029] Figure 4 A schematic diagram showing a possible design of reference DNA in the context of a point mutation assay;

[0030] Figure 5 A schematic diagram of template DNA used to explain the multiplex implementation of nested PCR is shown;

[0031] Figure 6 A schematic diagram of implementing quantitative real-time PCR in a microfluidic PCR array is shown.

[0032] Description of Embodiments

[0033] in accordance with Figure 1 Explain the design principle of the DNA template used, i.e., the target DNA 11 and the reference DNA 12. The classic TaqMan® system can be used as the basis of the PCR reaction batch, wherein, as described at the beginning, two different fluorescent probes are used. Here, the target DNA corresponds to the actual DNA sequence to be analyzed or to be quantified, such as the DNA sequence of a gene fragment. The target DNA 11 is supplemented with an artificial reference DNA, which has a specific sequence and is used in a specific amount. The target DNA 11 and the reference DNA 12 have the same primer binding sites, i.e., one binding site 13 for the forward primer and one binding site 14 for the reverse primer. These template DNAs 11 and 12 differ in the remaining base pair sequences 15 and 16. In particular, they have different binding sites 17 and 18 for the probes used. These two template DNAs 11 and 12 are also referred to as the quantifier 11 of the amplicon to be quantified and the artificial amplicon 12 of the artificial amplicon. The following table summarizes the design of the quantifier (target DNA) 11 and the artificial amplicon (reference DNA) 12:

[0034] .

[0035] The fluorophore of the fluorescent probe is selected so that the two colors can be distinguished from each other by means of a detector (filter set). It is appropriate that the orthogonal sequence 16 of the reference DNA 12 is different from the target sequence 15 of the target DNA 11. The GC content should be as identical as possible to the GC content of the target sequence 15 of the target DNA. The base pair lengths of the quantifier, i.e., the target DNA 11, and the artificial subunit, i.e., the reference DNA 12, should also be the same. As a result, the melting temperatures of the two amplicons 11 and 12 are very similar, so that in principle the same amount of amplification products are produced in an effective PCR. Quantitative real-time PCR is performed using these template DNAs, wherein the quantifier 11 and the artificial subunit 12 are amplified as a quasi-dual reaction in the same reaction vessel. During this period, the two probes are recorded, for example, after each PCR cycle or continuously. The artificial subunit 12 can be provided here in a predetermined amount within or above the detection limit, and must be detected as a signal of probe B in the case of successful PCR. In this case, the artificial subunit 12 is used as a reaction control. The amplification of the quantifier 11 and the gene fragment (target) to be quantified, which is optionally also present in the reaction batch, can be detected as a signal of probe A. The signals of probes A and B are now in a specific ratio. If there are the same starting amount of artificial sub-12 and quantifier 11, the two amplification curves are consistent. If there are more quantifiers 11, it is detected earlier, and the curve of artificial sub-12 is generated depending on its concentration. This can be calculated by the reaction efficiency and a fixed specific amount of artificial sub-12. The amount of artificial sub-12 provided is an absolute reference point here, which is known. The reaction efficiency can be determined by means of the curve shape of the exponential phase. The unknown starting amount of the quantifier 11 can thus be calculated using the absolute reference point.

[0036] Figure 2 The embodiment of the reaction system is shown in the case where the sample material (sample) is present as a genome. This can be used, for example, when a specific gene fragment is to be detected in a lysate. Figure 1 Compared to the principle of PCR, quantifier 11 (Q) and artificial 12 (A) are used here. In addition, the gene fragment 20 (S) to be amplified is located in the reaction batch as a cell lysate with genetic material formed by the genome of (one or more) cells. In this case, quantifier 11 and artificial 12 are provided in a predetermined amount in a ratio of 1:1. The selected amount can be, for example, close to above the detection limit. Another possible way is to match the amount to the ideal functional range of the PCR reaction so that the PCR is carried out particularly efficiently. Usually, various quantitative real-time PCR (qPCR) have limits within which the reaction is carried out effectively. Here, the detected C TThe value (which describes the starting point of the exponential growth of the curve) is in a linear ratio with the logarithmic starting amount used. If the amount of quantifier 11 and artificial 12 used is selected within this range, a signal should be detected in each case of successful PCR. The signal of artificial 12 is the signal that must be detected last in chronological order. If this signal is missing, the reaction control is negative. If the signal of quantifier 11 is detected simultaneously with the signal of artificial 12, this means that only quantifier 11 and artificial 12 are present in the reaction mixture, but no sample 20. This situation is Figure 2 1 and serves as a detection control for the principle function of the PCR batch. Here, lines 11 and 12 represent the fluorescence signals of the respective quantifier 11 (fluorophore A) and the artificial quantifier 12 (fluorophore B). If the same DNA fragment as quantifier 11 is present in the genome or in sample 20, this fragment from sample 20 is also highly amplified. This results in the signal of quantifier 11 being detected earlier ( Figure 2 B), wherein the detected signal is composed of the amplification of the quantifier 11 and the sample 20. Figure 1 As explained in principle, the starting amount of the DNA fragment to be tested of the sample 20 (sample) can now be calculated. Here, the predetermined amount of the artificial element 12 and the quantitative element 11 not only provides an absolute reference point for calculating the quantification, but also ensures the signal and serves as a control.

[0037] In another embodiment of the PCR process, the process can be carried out dynamically, which is achieved by making the signal of the quantifier 11 represent the termination criterion of the reaction so that the PCR process can be ended after the signal of the quantifier 11 appears. Since the amount of the quantifier 11 can be transferred to the effective range of the PCR process, the detection can be carried out roughly at the time midpoint of the planned process duration, that is, at the average number of cycles. In the case of a positive sample, that is, when the searched DNA fragment is present in the sample 20, the signal of the quantifier 11 (together with the signal of the sample 20) appears before the signal of the artificial quantifier 12, so the process time for measurement can be shortened. In this case, only qualitative description can be made after the reaction is terminated.

[0038] Figure 3A and 3B The concept is shown in the context of qualitative nested PCR (nested PCR). Such a PCR method can be used, for example, to detect mutations. For this purpose, the target region where the mutation is located is highly replicated from the genomic DNA of the cells. The ratio of wild type to mutation is then measured. For this purpose, a preamplification is performed before the actual detection reaction to ensure that there is enough material for the detection. This is particularly important when there is less cell material, for example in liquid biopsies with circulating tumor cells. Figure 3AAs shown, the concepts described herein are implemented in a system in which a mutation site 35 (target DNA) is first highly replicated in a sufficiently large fragment using a specific primer pair. On the genomic DNA of the sample 30, there are corresponding binding sites 33, 34 for the forward primer and the reverse primer for this first primer pair. In order to control, monitor and quantify the process, a probe binding site 37 of the first fluorescent probe A is selected adjacent to the primer binding site 33. A quantifier 21 is designed in accordance with the amplicon in the sample 30, namely the target DNA 21, which has corresponding primer binding sites 23, 24 and a corresponding probe binding site 27 for the fluorescent probe A. In addition, an artificial amplicon 22 (reference DNA) is designed that has the same primer binding sites 23, 24 and a different probe binding site 28 for the fluorescent probe B. These components 30, 21, 22 provide the basis for pre-amplification, which can be based on Figure 2 The principle of explanation is used for quantification. In addition, additional primer binding sites 43, 44 for another primer pair having a second forward primer and a second reverse primer are set for the subsequent detection reaction 102, wherein, in the genomic gene fragment 30, the binding site 43 for the second forward primer is connected to the binding site 37 for the fluorescent probe A. The binding site 44 for the second reverse primer is located downstream of the actual target DNA 35 (which represents the gene fragment to be detected). The corresponding primer binding sites 43, 44 are located on the reference DNA 22 (artificial subsequence for pre-amplification). On the target DNA 21 (quantifier for pre-amplification), different, i.e. orthogonal sequences are set at positions 143, 144 corresponding to the primer binding sites 43, 44 of the artificial subsequence 22. The sequence between the sequences 143, 144 on the quantitative subsequence 21 corresponds to the target DNA sequence 35 of the DNA fragment to be quantified of the sample 30.

[0039] After the pre-amplification 100 performed after adding the first primer pair, the amplified gene fragment 30' to be quantified (amplified sample) exists as a PCR product. In addition, there is an amplified artificial particle 22'. The amplified quantifier 21 also corresponds essentially to the amplified sample 30' in terms of sequence. In the primer binding sites 43, 44 for the second primer pair in the subsequent detection reaction 102, there is a binding site 47 for another fluorescent probe A' connected to the primer binding site 43 of the forward primer, which is used for the subsequent detection reaction 102. The artificial particle 22 or the amplified artificial particle 22' has another probe binding site 48 for another fluorescent probe B' after the primer binding site 43, which is also used for the subsequent detection reaction 102. An orthogonal sequence 26 is connected to the artificial particle 22 or the amplified artificial particle 22', which is orthogonal to the target sequence 35 of the sample 30 to be tested. Connected thereto are the binding site 44 of the reverse primer of the subsequent detection reaction 102 and the binding site 24 of the reverse primer of the preamplification. As explained above, the GC content and length of the base pair sequence between the artificial atom 22 and the corresponding fragments in the sample 30 and the quantifier 21 should be approximately equal.

[0040] The quantification of preamplification 100 is in principle as already described. Figure 2 as explained, and in Figure 3B . The signals from probes A and B are shown here respectively. Graph A shows the situation where the signal of the quantifier 21 (probe A) and the signal of the artificial sub 22 (probe B) overlap. In this case, there is no DNA fragment to be detected in the sample 30. Graph B shows the situation where the signal of the amplified quantifier 21 together with the amplified gene fragment from the sample 30 appears in time before the signal of the amplified artificial sub 22. In this case, the gene fragment sought is present in the sample 30. If the sample (sample) cannot be detected (graph A), the entire process can be stopped and it can be stated that no detection is performed (negative test result). If the sample is detected according to graph B, the PCR process can be continued until the artificial sub 22 is detected. Then, the process can be terminated optionally. Alternatively, a predetermined number of PCR cycles can also be performed. The efficiency can be calculated from the amplification curve of the sample 30 together with the quantifier 21. The final concentration of all amplicons and the starting concentration can be calculated with the help of the predetermined artificial sub 22. On this basis, a batch can be diluted and formulated with a new masterbatch mixture (step 101) so that it corresponds to the ideal input concentration for one or more subsequent detection assays (step 102). The dilution can be performed manually, for example if the reaction is performed in a batch system, such as a classical qPCR cycler. The process is preferably performed in a fully automated fluid processor, wherein microfluidic systems are particularly suitable. Here, the liquid can be diluted and distributed with the help of microfluidic pumping and aliquoting systems.

[0041] For the actual detection reaction 102, for example for point mutation detection, the reaction mixture is amplified with the primers required for this (second primer pair), and the signal trends of probe A' (curve 470) and probe B' (curve 480) are observed and evaluated. According to the reaction concept, the artificial sub 22' is in the minority, that is, the starting material of the artificial sub 22' present is less than the starting material of the sample 30'. This is because more sample amplicons are generated during preamplification 100, which are composed of amplified sample 30' and amplified quantifier 21. Therefore, it is very advantageous to dynamically terminate the PCR immediately after the detection, because in the exponential phase, the artificial sub 22' and the sample 30' make the estimation of the amount of amplicon more accurate than in the detection of the saturation phase. Since the artificial sub 22' is now present again in a specific amount and the sample 30' acts as a new quantifier, the second qPCR, that is, the detection reaction 102, can also be fully quantified. Therefore, the copy number from the beginning to the end of the process is known. Since the corresponding positions 143, 144 are selected to be orthogonal to, ie different from, the primer binding sites 43, 44 on the preamplified target DNA sequence 21, the amplified quantifier 21 of the first reaction (preamplification 100) is not considered in the second reaction (detection reaction 102).

[0042] For the detection reaction 102, another artificial molecule can also be mixed into its masterbatch mixture. Here, instead of the first pre-amplified artificial molecule, a new artificial molecule is added for the second detection reaction. This is useful because the first reaction mixture is usually diluted and the artificial molecule is detected thereby (but the added actual sample is not detected). Therefore, a specific amount of artificial molecule is added again after dilution for a more accurate determination. The other artificial molecule can, for example, be pre-stored in the (second) freeze-dried beads required for the detection reaction. This is particularly advantageous for determining the ratio of wild type and mutant types in point mutation detection. Another quantifier with the same primer binding sequence is also used. The amplicon of the first reaction 100 must then be diluted in such a way that it corresponds to the concentration of the second quantifier provided.

[0043] Figure 4An embodiment of a possible design of an artificial subunit (reference DNA) 52, 62 for point mutation detection is shown. Artificial subunits 52, 62 are provided for nested PCR in the context of point mutation determination. Generally, two general PCR detection strategies are used in point mutation detection. Here, mutation-sensitive primers (a) or mutation-sensitive probes or blockers (b) are selected. In method (a), primers are designed so that they can only bind in the presence of mutations. Such an example is the so-called ARMS (amplification-impeding mutation system) system. In method (b), mutation-sensitive probes or blockers that bind only in the presence of mutations are used (e.g., PNA-CLAMP system - peptide nucleic acid (PNA)-mediated PCR clamping; H.Ørum et al., Nucleic Acids Res. 21: 5332-5336, 1993). However, since these bindings are not 100% effective, a reference signal of the wild type is measured along with it. Therefore, for the implementation of the concept according to the invention, it is useful for such a detection to include a second quantity of subunits. For implementation, for artificial subunits 52 and 62, mutation 301 is introduced into the orthogonal sequence. Therefore, the binding site of the mutation should be included. Therefore, in the version 62 with mutation-sensitive primers, artificial subunit 62 includes the following fragments: binding site 23 for the first forward primer, binding site 28 for probe B, binding site 63 for the mutation-specific primer (which represents the forward primer of the second primer pair for the detection reaction 102), orthogonal sequence 26, binding site 44 for the reverse primer of the detection reaction 102, and binding site 24 for the reverse primer of the preamplification. For the detection system with mutation-sensitive blockers or mutation-sensitive probes, artificial subunit 52 is designed so that the binding site for the probe or the blocker includes the mutation site 301 at exactly the same position as in the mutant type. In other aspects, artificial subunit 52 corresponds to artificial subunit 62 or artificial subunit 22. With such a configuration, the signal of a 100% wild-type reaction (second quantifier) ​​and the signal of a 100% mutant (artificial quantifier 52 or 62) can now be measured in a reaction and compared with a sample. All this can be done in one reaction batch, which can greatly simplify full automation, for example in point-of-care applications. It is particularly advantageous if the corresponding masterbatch mixture is provided in lyophilisate form.

[0044] Figure 5Multiple implementations of nested PCR are shown, in which two detection reactions can be performed in one batch. In this embodiment, a lysate of a small number of cells, such as 10 to 1000 cells, is used. In the lysate, cells from enrichment, such as enrichment from circulating tumor cells or from immune cells such as enrichment of specific T cells, from body fluids such as blood, urine, spinal fluid or other body fluids may be present. These cells are lysed in a small volume and provided as a sample for performing the method. In the cell lysate, two gene fragments 70, 80 should be detected, i.e., for example, exon A (gene fragment 70) and exon B (gene fragment 80). First, gene fragments 70, 80 are amplified (pre-amplification) so that in subsequent steps, abnormalities of these gene fragments, such as mutations or functional typical gene sequences, such as genetic coding of antigenic epitopes, can be detected in one or more detection reactions. In the first step of pre-amplification, two target exons, i.e., gene fragments 70 and 80, are first amplified from the genetic material of the lysed cells. In addition, a sample control is performed. For sample control, a control exon C is amplified as gene fragment 90. For example, this can be an exon of the gene being searched for that does not have an abnormality. If it is amplified, the reaction is considered to be successful and proves that sample material, i.e., genetic material, is present in the sample. In order to detect and quantify the amplification of the control exon 90, target DNA (quantifier) ​​21 and reference DNA (artificial) 22 are used in the above-mentioned corresponding manner, and according to the above-mentioned principle, they match the control exon 90 in terms of structure and composition. If amplicons 70, 80, and 90 all have approximately the same length and the same GC content, approximately the same copy number should be produced for various templates in the reaction batch in the triple reaction. If there is a deviation in length and GC content, the amplified amplicons maintain a ratio, wherein the ratio can also be affected by DNA structure and epigenetic modifications. This means that even if the reaction efficiency may be low in the amplification of one of the exons, the ratio of the amplicon copies formed is still constant. This allows only one amplicon, i.e., the control exon C, to be measured quantitatively, thereby being able to determine the number of amplicons for all exons. Therefore, no complex three-probe design is required, but rather it is usually sufficient to quantify the control exon C (gene fragment 90) using only a two-probe system. Therefore, the preamplification is first quantified by the control exon 90, so that the amplification product for the subsequent detection reaction can be estimated as described above and optionally distributed and diluted in situ for optimal reaction conditions in the detection reaction. After distribution and dilution, a new master mix is ​​added, which is set for the specific assay of the detection reaction and can also be adjusted according to Figure 2 Quantitative description.

[0045] The design of various amplicons is preferably as follows: Exon A (gene fragment 70) has binding sites 71, 72 for primers for pre-amplification at the periphery of the amplicon. Exon B (gene fragment 80) and control exon C (gene fragment 90) have corresponding primer binding sites 81, 82 and 91, 92, but have different sequences. In exons A and B to be tested in the subsequent detection reaction, binding sites 73, 74 and 83, 84 for primers used in the second reaction (detection reaction) are respectively followed on gene fragments 70 and 80. If a probe is set for the detection reaction, its binding site is included in the sequence. Control exon C (gene fragment 90) has a binding site 97 for fluorescent probe A after primer binding site 91. Accordingly, according to Figure 2 As explained, for the quantification of the control exon C (gene fragment 90), a quantifier or target DNA 21 and an artificial quantifier or reference DNA 22 are supplemented, which are provided with the same primer binding sites 91, 92 as the control exon C (gene fragment 90). The target DNA 21 also has the same binding site 97 for probe A. The reference DNA 22 also has a probe binding site 98, but has a different sequence for binding to the fluorescent probe B. The number of copies N formed is inferred from the signal to be generated with this reaction batch. C and the starting amount N0. The amount of exons A and B (gene fragments 70 and 80) is calculated from the maintained ratio. Here, the ratio can be determined within the scope of assay development. This ratio is specific for the respective assay. This ratio is essentially constant, but must be measured, i.e. parameterized, for each application. Figure 2 The method explained above, the master mix for subsequent two separate and parallel-processable specific detections of exon A and exon B can each have a quantifier and an artificial quantifier, which have the same primer binding sites as the respective target exons A and B. For detecting point mutations, such as according to Figure 4 As explained, the quantifier can have a wild-type sequence, and the artificial quantifier can have a mutant sequence. Figure 5 The entire reaction process is schematically shown in the middle part of the figure. First, pre-amplification 200 is performed, wherein exon A, exon B, control exon C, quantification and artificial exon are present in the batch as templates. After the amplification reaction, the quantitative result 210 (N0 control exon C, N C By comparing exon C, N C , N0 exon A; N C In order to achieve optimal starting conditions for the subsequent detection reactions for exon A and exon B, in step 220, exon A (N S，2 Exon A) and exon B (N S，2Subsequently, by adding the masterbatch mixture for the respective detection reaction in step 230, for example, the respective specific mutation detection is performed, wherein for this purpose, in addition to exon A and exon B, the correspondingly designed quantifier A and artificial quantifier A as well as quantifier B and artificial quantifier B are added, such as Figure 5 This is described in the next section.

[0046] Figure 6 The implementation of the PCR concept in a microfluidic qPCR array 500 is shown. The array 500 is, for example, integrated in a chip consisting of structured silicon, wherein the array 500 is located in a microfluidic chamber provided with an inlet 501 and an outlet 502. In one possible embodiment, the individual reaction vessels of the array 500 can be controlled as a whole or liquid can be added thereto. For example, the pre-amplified sample can be flushed onto the array 500, thereby filling the individual reaction vessels of the array 500. The sealing can prevent the individual reaction vessels from being connected via diffusion in the second fluid step. If, for example, the individual reaction vessels of the array 500 are pre-soaked with a freeze-dried masterbatch and / or with primer and probe sequences, the method using the nxm array can achieve a maximum nxm multiple degree including quantitative and quality control. Preferably, the reaction batch according to the concept of the present invention can be developed based on the TaqMan® system. The synthesis of various template DNAs, particularly quantitative quantifiers and artificial quantifiers, can be performed using conventional nucleic acid synthesis. Preferably, the masterbatch containing the template DNA can be pre-stored as a lyophilizate. Micro-optical fluidic systems are particularly suitable for process automation.

Claims

1. A method for performing quantitative real-time PCR, characterized in that A PCR process is performed using at least one reaction mixture, wherein the reaction mixture provides a reaction batch for performing quantitative real-time PCR to quantify at least one DNA fragment, wherein the reaction mixture comprises - a specific amount of at least one target DNA corresponding to the sequence of the DNA fragment to be quantified, and - at least one reference DNA having a specific sequence and a specific amount that is not identical to the at least one target DNA, and - two different fluorescent probes with different sequences that generate signals at different wavelengths, and - Primers and deoxynucleotides and DNA polymerase, and wherein the target DNA and the reference DNA have the same primer binding site and different probe binding sites, and wherein one of the fluorescent probes is configured to bind to a target DNA fragment outside the primer binding site and another of the fluorescent probes is configured to bind to a reference DNA fragment outside the primer binding site; Characterized in that the GC content of the target DNA and the GC content of the reference DNA are identical within a deviation of at most 15%, and the base pair lengths of the target DNA and the reference DNA are identical within a deviation of at most 15%; and wherein a sample having DNA fragments to be quantified is added, and wherein fluorescent signals of at least two fluorescent probes are detected, and The test result is determined by the ratio of the signals of the fluorescent probes.

2. The method according to claim 1, characterized in that The target DNA and / or reference DNA and / or primers and / or deoxynucleotides and / or DNA polymerase are provided in lyophilized form.

3. The method according to claim 1, characterized in that The amount of reference DNA is present in a concentration corresponding to the detection limit of the DNA fragment to be quantified.

4. The method according to claim 1, characterized in that The reaction mixture is configured for detecting and quantifying DNA fragments from a genome, wherein target DNA and reference DNA are contained in a specific amount at a ratio of 1:1 in the reaction batch.

5. The method according to any one of claims 1 to 4, characterized in that The method is performed in a PCR array having a plurality of array containers.

6. The method according to any one of claims 1 to 4, characterized in that The method is used for a nested PCR process with preamplification and at least one subsequent detection reaction, wherein the amount of the preamplified PCR product is estimated by the quantification.

7. The method according to claim 6, characterized in that A first primer pair is used for pre-amplification, and at least one second primer pair is used for a detection reaction, wherein the target DNA and the reference DNA respectively have sequence fragments complementary to the primer sequences, and wherein the sequence fragment complementary to the second primer pair is located within a fragment between the sequence fragments complementary to the first primer pair.

8. The method according to claim 6, characterized in that The nested PCR process was used for point mutation detection.

9. The method according to claim 6, characterized in that The nested PCR process is a multiplex process for detecting at least two specific gene fragments in a genome, wherein, in order to quantify the pre-amplification, a control reaction is performed, in which a control exon is amplified from the genome, and wherein target DNA and reference DNA are designed to match the control exon, and wherein the amount of amplification of the gene fragment to be detected in the pre-amplification process is inferred from the quantification of the amplification of the control exon.

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