How to perform real-time PCR

Abstract

In a method (10) for carrying out a nucleic acid amplification process, a sample nucleic acid and a reference nucleic acid are amplified in separate reaction batches, and an amplification signal is observed in real time. The number of amplification cycles and / or the duration of the amplification process are dynamically adjusted according to the amplification signal.

Description

[Technical Field]

[0001] The present invention relates to a method for performing real-time PCR, in which PCR cycles are performed that amplify a sample nucleic acid and a reference nucleic acid. Furthermore, the present invention relates to a computer program configured to perform this method. [Background technology]

[0002] Polymerase chain reaction (PCR) is a sensitive bioanalytical method for detecting specific gene fragments or, in general, nucleic acid sequences. Here, specific DNA sequences are replicated or amplified by cyclic replication. Replication requires the enzyme DNA polymerase. The products of one replication cycle serve as starting material or as templates for the next replication cycle. A known embodiment of PCR is so-called real-time PCR, in which the course of the reaction can be followed, in particular by fluorescent probes. Real-time PCR makes it possible to quantify the initial amount of DNA present in the reaction mixture before amplification. Quantification is performed in each reaction based on a reference measurement, which is processed and measured together in separate reaction batches in parallel.

[0003] The polymerase chain reaction proceeds through multiple amplification cycles. First, the original DNA is denatured, where it is separated into its individual strands (melted). In this state, primers can bind (anneal) to the individual strands in the next step. In the subsequent step, DNA polymerase binds and synthesizes each opposing strand of DNA in one direction, starting from the bound primer (extension). After this first amplification cycle, the primers are denatured again and recombine, followed by further synthesis of the opposing strand. Therefore, the reaction batch must contain DNA molecules as templates, primers, nucleotides, and the enzyme DNA polymerase. Denaturation, primer hybridization, and extension are controlled by adjusting the temperature. Therefore, the PCR process is usually carried out in a thermal cycler, where approximately 20 to 50 amplification cycles are typically intended, with the number of each amplification cycle being preset.

[0004] German Patent Application Publication No. 102010052524 describes a PCR method for qualitatively and quantitatively detecting nucleic acid sequences in real time, using a fluorophore-labeled DNA probe. A mixture of double strands bound to labeled primers is generated by the primer under hybridization conditions. By adding a polymerase with exonuclease activity, the labeled DNA probe is cleaved, quenching is stopped, and a measurable fluorescent signal is generated.

[0005] Disclosure of the Invention Advantages of the invention The present invention provides a method for carrying out a nucleic acid amplification process, in which a sample nucleic acid and a reference nucleic acid are preferably amplified in separate reaction batches. According to the present invention, the amplification signal is monitored in real time, and the number of amplification cycles and / or the duration of the amplification process are dynamically adjusted depending on the amplification signal. To monitor the amplification signal, the signal can be detected in a manner known per se, preferably using a fluorescent probe to make the resulting amplification of the nucleic acid detectable. Here, the system can be configured so that the fluorescence increases proportionally to the amount of amplified product, and various fluorescent dyes can be used. For example, DNA dyes such as cyanine dyes (e.g., SYBR® Green or PicoGreen®) can be used, which are embedded in double-stranded DNA. Another possibility is the use of so-called FRET probes (Förster Resonance Energy Transfer: FRET) in which a donor fluorescent dye interacts with an acceptor fluorescent dye. F oerster- R esonanz e nergie t The detected and evaluated amplification signal is compared with a control, and based on this, the number of amplification cycles and / or the duration of the amplification process are dynamically adjusted depending on the amplification signal. That is, the essence of the present invention is to detect and evaluate the amplification signals of the sample and the reference or control in real time or at several points in the process, and based on this, to perform a predefined action by dynamically adjusting, in particular, the number of amplification cycles and / or the duration of the amplification process.

[0006] Preferably, this process is real-time PCR, in which PCR cycles are performed to amplify the sample nucleic acid and the reference nucleic acid. In this preferred embodiment, the cycles whose number is dynamically adjusted are PCR cycles. Preferably, an amplification signal is determined for each PCR cycle performed. Thus, for example, after each PCR cycle, a signal can be detected and evaluated. Therefore, based on this so-called PCR simultaneous evaluation, for example, after each cycle, it can be determined whether a new cycle should be started or the entire PCR process should be stopped. For example, PCR can be stopped when a signal increase is confirmed in the sample using the sample nucleic acid and / or in the batch using the reference nucleic acid. Therefore, a cycle threshold (C ) that represents the start of exponential increase in the amplification signal is determined. T The ability to terminate the process after detection of a certain value (value) can shorten the PCR process time. It is therefore even possible to stop the PCR once a defined and known amount of PCR product has been produced.

[0007] Thus, it is possible to achieve consistent production of the same amount of product during amplification, despite variations in PCR conditions due to, for example, different properties of the DNA-containing samples.

[0008] Furthermore, the method according to the present invention is also suitable for other amplification processes using DNA polymerases (amplification enzymes), such as whole genome amplification (WGA) or other DNA amplification methods, particularly isothermal DNA amplification methods in which the amplification process proceeds at a substantially constant temperature. These processes may use, for example, a combination of various polymerases, helicases, ligases, or enzymes from a DNA replication ensemble. In these embodiments, the duration of the amplification process is dynamically adjusted according to the amplification signal.

[0009] It should be understood that observing the amplification signal in real time does not necessarily mean that the signal is detected continuously, but rather at specific time-discrete points assigned to, for example, an individual PCR cycle, e.g., the signal may be detected after each binding step or each extension step of a PCR cycle.

[0010] In this context, nucleic acid should be understood to mean, in particular, DNA, which serves as a template for amplification. Both the sample nucleic acid and the reference nucleic acid or comparative sample are processed together in separate reaction batches. Here, the reaction batch contains each nucleic acid as a template DNA. In addition, the reaction batch contains, for example, the usual reagents for a PCR batch, in particular, primers that interact with a single strand of DNA at a specific position through a complementary nucleotide sequence to determine the starting point of DNA synthesis. In addition, a thermostable DNA polymerase and deoxyribonucleoside triphosphates as components of the DNA strand synthesized by the DNA polymerase are included. In addition, ions necessary for the DNA polymerase to function and an appropriate buffer are present. In the case of other amplification processes, particularly isothermal amplification processes, in which this method can also be used advantageously, corresponding components that are also known per se are included in the reaction batch.

[0011] This method may be intended to begin real-time observation and / or evaluation of the amplification signal only after a presettable minimum number of amplification cycles and / or a presettable minimum duration of the amplification process has been performed. For example, this minimum number of cycles may be defined so that a signal is not expected before this number of cycles or minimum period of amplification has elapsed. This configuration has the advantage of saving capacity for observing and evaluating signals during phases when no relevant results are expected. The minimum number of PCR cycles may be, for example, in the range of 10 or less. For example, during these initial cycles, a basic baseline may be generated for subsequent evaluation.

[0012] In a preferred configuration of this method, the process is terminated when the signal intensity of the amplification in the batch using the sample nucleic acid reaches and / or exceeds the signal intensity of the amplification in the batch using the reference nucleic acid. In this case, the amount of sample nucleic acid is assumed to correspond to the amount or concentration of the reference nucleic acid. By configuring the method in this way, it is possible to particularly determine the initial concentration of the sample nucleic acid and then terminate the process. Stopping the process before reaching a presettable maximum number of amplification cycles or before the presettable maximum duration of the process has the particular advantage of minimizing the occurrence of undesired by-products (such as primer-dimer formation), which may occur especially at the end of the PCR reaction when the number of cycles is high. This facilitates more detailed analysis in the further characterization of the amplification product, if necessary.

[0013] The amplification process can be stopped after the maximum number of amplification cycles and / or the maximum duration of the process that can be preset as needed, whereby no significant increase in the amplification signal in the batch using the sample nucleic acid has been observed up to this point. This maximum number can be, for example, the number of PCR cycles selected in conventional PCR experiments, for example, 50 PCR cycles.

[0014] Overall, the method described herein does not require new assay development, since conventional reagents and reaction parameters are used in the amplification process. Depending on the application, only the process control, particularly the process duration and, for example, the number of PCR cycles, and possibly the dynamic intervention of the composition of controls, are newly relevant to the system. Here, the described method allows for the controlled and complete automation of the assay workflow, without the need for an intervening quantification method, which may require sample collection and subsequent purification of the amplification product.

[0015] This method can be performed, for example, by observing an amplification signal for each amplification cycle performed. If the signal significantly increases, each cycle is classified as "amplification." Comparison of this classification result between the batch using the sample nucleic acid and the batch using the reference nucleic acid for each cycle is used in the evaluation. Instead of (or in addition to) individual amplification cycles, signals can be set for configurable time points during the process, and the signals are detected at these configurable time points. For example, signals can be collected at a rate of 1 second to 1 minute, i.e., signals can be detected (e.g., by taking fluorescent images) every 1 second, 30 seconds, or 1 minute, and evaluated, for example, as described above. Depending on the application, the observation time range can be, for example, 1 second to 10 minutes, preferably 30 seconds to 5 minutes. In a particularly preferred configuration of this method, the results of the amplification process are evaluated as an index vector notation. In this regard, amplification cycles or time points classified as "amplification" can be assigned, for example, an index value of "1," and other cycles or time points can be assigned an index value of "0."

[0016] This method allows the initial amount of sample nucleic acid to be determined and / or controlled in a particularly advantageous manner. For this purpose, it is preferable to process at least two comparison samples together in parallel, preferably with a defined, i.e., known and predetermined, initial amount of reference nucleic acid. For example, a comparison sample with a minimum initial amount or minimum initial concentration and at least one comparison sample with a maximum initial amount or maximum initial concentration can be used. In this case, the maximum initial amount (maximum standard concentration) and the minimum initial amount (minimum standard concentration) can be used to adjust the detection range. Additional comparison samples with concentrations within this range allow interval assignment for the initial concentration of the sample and create multiple subintervals that can be used, for example, for quality control. Different concentrations of comparison samples or standard samples can differ, for example, by 10 times. As soon as an amplification signal is visible in the sample (index value "1"), the amplification process is stopped, and the initial concentration or concentration interval in the sample can be estimated by comparing it with each index value reached up to that point in the batch with the standard concentration. A particular advantage here is that the time required to carry out the process can be shortened. There is no need to perform the maximum number of cycles or maximum process duration required for conventional methods to detect amplification and its amount, and the process can be performed at a cycle threshold (C) that marks the onset of exponential growth of the amplified signal. T The associated time savings are particularly advantageous when used in point-of-care (PoC) applications.

[0017] In another preferred embodiment of this method, this method is used for infectious disease detection.Here, at least one comparison sample (for example, a characteristic gene fragment of a pathogen) having a concentration of the nucleic acid to be detected that represents the lower limit of detection is processed together.This detection limit can be the latest stopping criterion for the amplification reaction.If a signal is detected early in the batch using the sample nucleic acid, especially if an "amplification" signal is detected, the test can be evaluated as positive.Here, additional comparison samples with different concentrations of the nucleic acid to be detected can be processed together, and in an effective test, the time sequence of the appearance of the amplification signal in the comparison sample should correspond to the sequence of concentrations.

[0018] In another configuration of this method, this method is used as mutant detection.For this purpose, a comparison sample with a defined concentration of the corresponding nucleic acid, which has 100% proportion of the mutant to be detected, and another comparison sample with a defined concentration of the nucleic acid (wild type), which preferably has 0% proportion of the mutant to be detected, are processed together.Between these two limits, multiple mixing ratios of mutant nucleic acid and wild type nucleic acid can be selected and used.

[0019] In a further configuration of this method, this method can be used for whole genome amplification (WGA).A particular advantage here is that the amount of amplification product produced can be controlled by processing a corresponding comparison reaction with a known nucleic acid concentration.In particular, in the case of whole genome amplification, the problem of undesired by-products may occur, especially when the number of cycles is high or after a relatively long amplification period, i.e., at the end of the WGA process.In contrast, the method described herein has the advantage that the process can be stopped as soon as a certain product amount or product concentration is reached, thereby preventing or minimizing the formation of undesired by-products.

[0020] When using a method for whole genome amplification, it is preferable to process at least one comparison sample containing a defined concentration of nucleic acid (DNA) of the reference genome. This first comparison sample is preferably specific for each species. For example, if a human genome is to be amplified, the DNA of another human, or preferably a mixture from multiple different humans, can be used as the reference genome, thereby taking genetic diversity into account. The defined concentration or amount of the reference genome preferably corresponds to the maximum usable amount of DNA in the whole genome amplification system. Furthermore, it is preferable to provide a second comparison sample that does not contain the nucleic acid to be amplified (no template control). Furthermore, it is preferable to provide a so-called quantity reference as a third comparison sample containing a defined amount of nucleic acid of the reference genome, and this defined amount corresponds to the desired target amount of product in whole genome amplification. Here, this batch of the third comparison sample does not contain an amplification enzyme. That is, no amplification occurs in this third comparison sample during the process. In the case of this third comparative sample, by using a fluorescent dye that is embedded in double-stranded DNA, i.e., independent of the amplification that occurs, the fluorescent dye is embedded in the already existing double-stranded DNA, so that the resulting fluorescent signal corresponds to the signal that should be achieved by processing the original sample in whole genome amplification. The occurrence of the amplification signal in the comparative sample compared to the signal in this sample defines various checkpoints that allow the process to be carried out in a controlled and automatable manner.

[0021] In a further embodiment of this method, the method is used for targeted or controlled preamplification, for example, in the field of nested PCR. Here, the amount of amplified nucleic acid or PCR product is controlled and adjusted by processing a corresponding standard together. The method described herein can also be used for nested PCR using a first multiplex PCR and at least one second singleplex PCR, whereby the amount of nucleic acid amplified in the first multiplex PCR can be controlled. In general, in nested PCR, multiple predefined gene fragments are amplified in a first multiplex PCR. Then, in one (or more) second singleplex PCRs, individual genes or gene fragments are specifically detected based on the first PCR products. For example, this method can be used for mutant detection, where gene fragments in which the mutant(s) to be detected are potentially located are replicated. Only then are individual mutants specifically detected in a second reaction. However, these second reactions often have a limited ideal operating range. This means that too little or too much input material from the first PCR can adversely affect the efficiency of the reaction. The method described herein allows for the measurement of how much sample starting material was present in the first PCR. Furthermore, the amount of amplification or PCR product generated in the first reaction can be controlled by stopping the reaction when a specific target value is reached. Based on the detectable and controllable concentration of the PCR product in the preamplification, the corresponding dilution of the first PCR product can then be adjusted, thereby adjusting the PCR product from the first reaction used as template DNA in the second reaction to an optimal concentration for the subsequent detection reaction.

[0022] The described method is particularly suitable for implementation in microfluidic systems, for example as lab-on-a-chip systems, whereby very small sample volumes are advantageously required, whereby the advantages of the described system are particularly apparent in relation to the possibility of automation.

[0023] The various components for carrying out the described methods may be provided to the user, for example, as a kit, which may include, among other things, comparison samples, reagents, enzymes, and buffers required for each process.

[0024] The method may be implemented as a computer program configured to perform the method, which may be stored on a machine-readable data carrier and / or implemented in a corresponding control device for performing the amplification process.

[0025] Further features and advantages of the invention will become apparent from the following description of exemplary embodiments in conjunction with the drawings, in which the individual features can be realized either individually or in combination with one another. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a schematic diagram of the steps of a real-time PCR implementing the method according to the invention. [Figure 2] FIG. 1 is a diagram of the evaluation of the fluorescent signal in a PCR process within the scope of the method according to the invention. [Figure 3] 1 is a diagram of the implementation of an amplification process according to the method according to the invention for determining the initial concentration of a sample; [Figure 4] 1 is an evaluation of the amplification process by the method according to the invention in the implementation of infectious disease detection using an index vector notation. [Figure 5] 1 is an evaluation of the amplification process by the method according to the invention in the performance of mutant detection using an index vector notation. [Figure 6] FIG. 1 is a flow diagram of a method according to the present invention for whole genome amplification. [Figure 7] 1 is a schematic diagram of the equipment components required to perform real-time PCR according to the method of the present invention.

[0027] Description of the Examples FIG. 1 shows a schematic flow diagram of real-time PCR 10 according to the method of the present invention. After the PCR process starts at 11, PCR cycles are initiated, with the individual steps being controlled by controlling the temperature in the thermal cycler. At regular intervals, particularly at defined time points within the PCR cycle (or depending on specific time points in the isothermal amplification process), amplification signals are detected and evaluated, e.g., as fluorescent images. The selection of appropriate time points may depend, for example, on the probes used. In the example shown here, measurements are performed after each binding step. However, in most cases, measurements are performed after each extension step. Each PCR cycle includes step 12, in which template DNA is denatured. The template DNA used is a sample nucleic acid and a reference nucleic acid in separate reaction batches. After denaturation step 12, the primers are bound (annealed) in step 13. Subsequently, in this example, the amplification signal is measured in step 14. The measured signal is evaluated in step 15, specifically to determine whether the measured signal is classified as "amplification." It is then decided whether further PCR cycles should be performed, particularly by comparison with a reference sample. For example, if it is determined in step 15 that the measured signal is classified as background, i.e., not "amplification," the PCR cycle continues from extension step 16. A new PCR cycle then resumes from denaturation step 12. However, if it is determined in step 15 that the measured signal is not assessed as background but is classified as "amplification," the PCR process can be stopped, whereupon further analysis and evaluation of the formed PCR products can be performed, if desired (step 17).

[0028] The detection of the signal in step 14 is based on fluorescent probes that make it possible to detect amplification that occurs by various per se known techniques, for example by incorporation during DNA synthesis or by binding or embedding into DNA. It is then tested, especially statistically, whether this new data point can be classified as background according to the data points already measured in the previous PCR cycles, or whether the signal deviates significantly from the previously determined background and can be called "amplification".

[0029] For PCR processes, it is convenient to predefine minimum and maximum PCR cycle numbers as boundary conditions. The minimum cycle number defines the earliest signal is expected after this. These data points are automatically assigned to background and are not tested for amplification. This minimum cycle number can be set, for example, to 10 or less. A basic baseline can be generated during these initial cycles. If no amplification is detectable in the sample, the maximum cycle number can serve as a stopping criterion. This number is also predefined for conventional PCR processes.

[0030] Figure 2 illustrates the evaluation of the detected fluorescence signals (step 15 in Figure 1). These fluorescence signals can be detected for individual PCR cycles or at specific time points during the amplification process, particularly in the case of isothermal amplification processes (e.g., in the case of whole genome amplification). Subfigure A shows the background (BG) or a baseline formed by individual data points (white circles) measured particularly in early PCR cycles where amplification is not expected. The framed lines around the individual data points represent an estimated background with specific tolerance values. This background, thus defined, serves as the basis for testing subsequent data points based on the fluorescence signals measured in subsequent PCR cycles or subsequent amplification processes. Subfigure B shows, as black circles, the data points to be measured later that are present within the framed lines of the background, based on further fluorescence signals in subsequent PCR cycles or subsequent processes. The latest data point indicated by a cross represents the current measured value, which also lies within the framed lines of the background. Here, it is assumed that no amplification has occurred. Thus, first an old background is calculated based on the data points from previous cycles. That is, the background for all points except the current measured value (BG1) is determined. If the current data point is present, a second background BG2 is calculated, including the current data point. Now, it is possible to statistically test whether the two possible backgrounds BG1 and BG2 are significantly different. The statistical evaluation can be carried out according to the following rules: Hypothesis H1: BG1 = BG2 Hypothesis H0: BG1 ≠ BG2 As in Subfigure B, if P(H1) > P(H0), there is no significant difference and no amplification has occurred. The amplification process is continued. In contrast, if the background is significantly changed by the current data point (P(H1) < P(H0)), amplification is assumed as illustrated in Subfigure C. This information is the basis for further steps in the amplification process and can lead to the termination of the process.

[0031] Figure 3 illustrates the implementation of an amplification process for determining the initial concentration of sample DNA. This example is illustrated using a PCR process. This and subsequent examples can also be applied, for example, to isothermal amplification processes, where the observed amplification signal is assigned to individual time points in the amplification process rather than to individual PCR cycles. In parallel with sample 31, various reaction batches with standards 32, 33, 34, and 35 are processed together as comparison samples. Here, standard 35 represents the maximum standard concentration S1, and standard 32 represents the minimum standard concentration S4. In principle, many intermediate levels of standard concentration can be arbitrarily selected between the maximum and minimum standard concentrations. In this example, there are two concentrations, S2 and S3. S1-S n The number of different standard concentrations determines the resolution of the concentration measurement. After the start of the PCR process 30, all batches are run in parallel, and during each PCR cycle, amplification signals are detected in step 36. In step 37, whether amplification has occurred is evaluated. This can be done, in particular, using the method described in connection with Figure 2. The numbers 1 and 0 shown in area 38 represent classification as amplification ("1") or no amplification ("0"). If amplification is confirmed in sample 31, the PCR process can be stopped. The amplification results for standard samples 32-35 can then be compared to derive the concentration interval in which the initial DNA concentration in sample 31 fell. If amplification is not confirmed in sample 31 but occurs already at the minimum standard concentration 35, the PCR process can also be stopped because the initial concentration of sample 31 is below the detection limit defined by the minimum standard concentration 35. This procedure is realized by query 39, which determines whether amplification is confirmed in either of the two batches between sample 31 and comparison sample 32 or the standard with the minimum concentration. In this case, the PCR process is terminated (step 40). If no amplification is observed in either sample 31 or standard S4 with minimum concentration 32, the next PCR cycle is performed in step 41. This method allows unambiguous allocation of concentration intervals, where the concentration intervals are defined by the number of standards. Here, standards S1 to S nis expected to send amplification signals continuously from the maximum concentration to the minimum concentration according to the progress of the PCR process. Amplification was confirmed in sample 31, and at the same time, standards S1 to S i (i < n), if amplification is confirmed, the initial concentration of sample 31 is [S i , S i+1 intervals. If the confirmation of amplification in the standard does not match its concentration order, there is no valid test. That is, if a batch with a smaller standard concentration shows amplification in the PCR cycle and a standard with a larger concentration still does not show amplification, the reaction is not at the same efficiency or is not equivalent. For example, the standard concentrations can be selected such that they differ from each other by a factor of 10. This corresponds to quantification in the sense of conventional real-time PCR.

[0032] Figure 4 illustrates a method using an index vector notation for infectious disease detection. In addition to the original sample 51, three standards 52, 53, and 54 are processed together, where standard 52 is standard S1 with the lowest concentration of DNA to be detected, standard 53 is standard S2 with a medium concentration of DNA to be detected, and standard 54 is standard S3 with the highest amount of DNA to be detected. Standard S1 represents the detection limit, which is the current stopping criterion for the reaction. If amplification is confirmed early in sample 51 and the order of amplification in the standards corresponds to their concentration order, the test is evaluated as positive. Figure 4 summarizes the evaluation of whether a reaction in a particular PCR cycle should be evaluated as an amplification in an index vector. In this vector, each reaction vessel or each PCR batch (sample and standard) has inputs that are newly evaluated after each cycle. If a signal above background is detectable, the reaction is evaluated as "amplification." In the index vector, this is assigned an index value of 1 (true). If no amplification is confirmed, the reaction index is set to 0 (false). In this example, standard S3 is the largest standard and is listed on the left as the upper detection limit. A second standard S2, which is quantitatively between the largest and smallest standard, follows next. The smallest standard S1, representing the detection limit, follows in the third position. The original sample 51 follows as the last entry in the state vector. The experiment is initialized with / = [0,0,0,0]. Figure 4 shows four vectors representing valid tests. All other 12 possible cases are not allowed, and the test should be reported as invalid. If / = [1,1,1,0], the signal is within the detection limit. In this case, one or more cycles can be added as needed to ensure that small differences between individual reaction vessels do not cause errors in the test interpretation due to noise in reaction efficiency. The last column in the figure shows the test result for each vector, either positive (+) or negative (-). As soon as one of these vectors is present, the reaction can be stopped.

[0033] FIG. 5 also illustrates the implementation of the method for mutant detection applications, also represented as an index vector. For mutant detection, a predefined amount of sample DNA is typically used in the sample 61 to be tested. Because this amount is predefined, the same amount of standard or reference DNA is always used for standards 62, 63, and 64. Standard S1 64 contains pre-inserted template DNA, 100% of which contains the mutant (M) to be detected. This standard S1 forms the upper limit for the initial amplification detection. The lower limit, and thus the final termination criterion for the reaction, is standard S3 62, which contains 100% wild-type template (W). Between these two limits, multiple mixing ratios of mutant and wild-type DNA can be selected. In this example, another standard S2 63 is provided, containing 50% mutant (M=50%). By adjusting the mixing ratio of mutant and wild-type DNA, the sample can be divided into percentage bins, similar to a histogram. The standard S2 selected here with M=50% allows the proportion of mutants to be classified as greater than or less than 50%. Thus, for example, the ploidy of genes can be determined for this batch. By adding additional standards and estimating the efficiency numerically, finer refinement can be achieved. As explained in the previous example with reference to Figure 4, the reaction is controlled via a state vector, and test decisions are made accordingly.

[0034] Figure 6 illustrates a method related to whole genome amplification. In whole genome amplification, all sequences occurring in a sample are amplified; that is, not only defined DNA sequences addressed by primers are amplified. Conventional whole genome amplification does not use fluorescent probes, as in typical real-time PCR. Instead, the generated amplification products are visualized and quantified using specialized dyes. These specialized dyes (e.g., PicoGreen®, SYBR® Green) emit more light when embedded in double-stranded DNA, so an increase in fluorescence indicates that amplification has occurred. That is, if a fluorescent signal is detectable, this indicates the presence of double-stranded DNA, allowing amplification to be evaluated. The dyes are added in defined amounts to the reaction mixture of the process batch. In the whole genome amplification process, in addition to the original sample 71 (S), three additional comparison samples 72, 73, and 74 are processed together. The comparison sample 72 does not contain template DNA as a so-called no-template control (NTC). Since there is no DNA to be amplified, no amplification should be observed in this sample. The DNA of a reference genome (RG) is also processed as a further comparison sample 73. This reference genome is advantageously species-specific. For example, if the entire human genome is to be amplified, the reference genome is one or more other human genomes. The amount of reference genome used corresponds, for example, to the maximum amount available in a suitable whole genome amplification system. Therefore, this reference genome of the comparison sample 73 should first send an amplification signal. After start 70, reactions are performed and tested for amplification until the reference genome 73 and sample 71 are positive for the state vector (state 75). That is, at state 75, both the reference genome 73 and sample 71 show amplification, and the NTC control 72 shows no reaction or amplification. Only at this state is sufficient DNA present in the sample, and no nonspecific primer amplification (primer dimer formation) has occurred up to this state, as demonstrated by control 72. This state represents the first checkpoint.Once the first checkpoint condition is met, the reaction continues, but a new stopping criterion 76 is tested. The new test criterion 76 is a comparison of the amplification intensity of the sample 71 and the quantity reference 74. This quantity reference 74 contains the desired amount of reference genome, which corresponds to the desired amount of product in whole genome amplification. Here, the quantity reference 74 contains all components in the WGA batch, as in other batches, except for the amplification enzyme. As a result, no amplification occurs in the quantity reference 74 during the reaction; i.e., no DNA synthesis occurs. Only a reference fluorescent signal is emitted by the pre-inserted fluorescent dye. If the amplified sample 71 and the quantity reference 74 have the same fluorescent intensity, it can be assumed that essentially the same amount of double-stranded DNA is present in both batches, and the reaction can be stopped in step 77. An additional stopping criterion may be that the NTC control 72 indicates an amplification signal.

[0035] This method can also be applied to targeted specific preamplification, in which the amount of DNA synthesized in preamplification is controlled. Here, instead of the entire genome, specific primers are used, thereby correspondingly highly copying specific gene fragments. Here, instead of dyes embedded in double-stranded DNA, specific fluorescently labeled probes that generate fluorescent signals depending on the DNA synthesized, such as TaqMan® probes with fluorophores and quenchers, can also be used as probes. The quantity reference includes the desired target amount of amplicon, the corresponding amount of cleaved probe, i.e., the same amount of free fluorophore and quencher, and the complementary remaining amount of probe. This is based on the fact that a defined initial amount of probe (N0=c0V) is pre-defined in a real-time PCR batch in the TaqMan® probe system. When amplification begins, the probe is cleaved. The amount of probe and free fluorophore is determined by the resulting copy number N アンプリコン The remaining probes N s is N s =N0-N アンプリコンInstead of an NTC control, an additional reference is used as a stopping criterion, which allows finding the detection limit of amplification (LoA - amplification limit). Here, the minimum genome dilution to be used is used. Thus, the first checkpoint here is the amplification time point at which the assay-specific predefined genome dilution, i.e., the reference LoA, is amplified.

[0036] The method of whole genome amplification according to the description of Figure 6 and the method of variant detection according to the description of Figure 5 may be combined with each other and configured as a controlled workflow for variant detection. Here, microfluidic systems and / or pipetting robots may be used. Such a fully automated workflow may provide very advantageous usability of the method according to the present invention, especially in connection with microfluidic systems that may be used, for example, in point-of-care applications.

[0037] FIG. 7 illustrates device components that can be used for the described real-time PCR process. The basis here is a device that enables optofluidic real-time PCR, i.e., allows for signal reading of optical signals, so that amplification in individual samples or PCR reactions can be monitored using fluorescent signals. Such a device comprises a heating and cooling system 101 (thermal cycler) that interacts with various PCR reaction batches 102. Furthermore, the device has an optical unit 103 that reads the amplification signal. Furthermore, a device 104 for fluid processing, such as a robotic system or a corresponding microfluidic system, may also be provided. Overall, such a system is advantageously configured as a microfluidic system, since microfluidic systems can operate with very small sample volumes and allow partial or complete automation. Furthermore, the system is equipped with a reaction control unit 105 that evaluates the optical data on the fly. To realize the feedback real-time PCR system of the present invention, the reaction control unit 105 is configured to interact with all units of the system. In particular, the reaction control unit 105 can be controlled to dynamically adjust the number of PCR cycles depending on the observed amplification signal.

Claims

1. 1. A method for performing a nucleic acid amplification process, wherein a sample nucleic acid and a reference genome are amplified in separate reaction batches, comprising: The method is carried out in a microfluidic system; the microfluidic system includes an optical unit and a reaction control unit; the optical unit is an optical unit that reads an amplified signal, The reaction control unit monitors the amplification signal in real time and adjusts the number of amplification cycles and / or the duration of the amplification process based on optical data from the optical unit, The method is used for whole genome amplification, wherein at least one first comparison sample having a defined nucleic acid concentration of a reference genome is processed together, and the amount of the reference genome used corresponds to the maximum amount of DNA usable in the whole genome amplification system; A method characterized in that a second comparison sample without the nucleic acid to be amplified and a third comparison sample having a defined amount of nucleic acid of a reference genome are additionally processed together, wherein the defined amount of the third comparison sample corresponds to the desired target amount of amplification product in the whole genome amplification, the reaction batch of the third comparison sample does not contain an amplification enzyme, and the amplification signal is based on the use of a fluorescent probe, preferably a fluorescent dye or FRET probe embedded in double-stranded DNA.

2. 2. Method according to claim 1, characterized in that said amplification of nucleic acids is carried out in the field of real-time PCR and said amplification cycles are PCR cycles.

3. 3. The method according to claim 1, wherein the observation and / or evaluation of the amplified signal begins in real time once a predefinable minimum number of amplification cycles and / or a predefinable minimum duration of the amplification process has been carried out.

4. 4. The method according to claim 1, wherein the amplification process is stopped after a predeterminable maximum number of amplification cycles and / or a predeterminable maximum duration of the amplification process has been performed.

5. 5. The method of claim 1, wherein the amplification signal is observed for each amplification cycle performed and / or for a configurable time point, and if the signal increases significantly, each cycle or each time point is classified as "amplification," and the evaluation uses a classification comparison between the sample nucleic acid and the reference genome.

6. The method of claim 5, wherein the results of the amplification process are evaluated as an index vector representation, wherein cycles or time points classified as "amplification" are assigned an index value of "1" and other cycles or time points are assigned an index value of "0".

7. 7. The method according to claim 6, characterized in that said method is used for the control of amplification.

8. A computer program for controlling a nucleic acid amplification process, characterized in that the computer program is configured to carry out the method according to any one of claims 1 to 7.

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