PROCEDURE FOR PERFORMING REAL-TIME PCR

Description

[0001] The present invention relates to a method for performing real-time PCR, wherein PCR cycles are carried out for the amplification of sample nucleic acids and reference nucleic acids. A computer program configured to perform the method is further disclosed. State of the art

[0002] The polymerase chain reaction (PCR) is a sensitive bioanalytical method for detecting specific gene segments or nucleic acid sequences in general. In this process, specific DNA sequences are amplified or replicated through cyclic duplication. The enzyme DNA polymerase is required for this replication. The products of a replication cycle serve as starting materials or templates. (template) for the next amplification cycle. A well-known implementation of PCR is so-called real-time PCR, in which the reaction progress can be monitored, particularly using fluorescent probes. Real-time PCR allows the quantification of the initial amount of DNA present in the reaction mixture before amplification. Quantification is based on reference measurements, which are carried out and measured in parallel in separate reaction mixtures during each reaction.

[0003] Polymerase chain reactions proceed in several amplification cycles. First, the starting DNA is denatured and separated into its single strands ( melting ) . In this state, primers can then attach to the individual strands in the next step ( annealing ) . In the next step, the DNA polymerase binds and synthesizes the respective opposite strand of DNA in one direction, starting at the bound primers ( elongation ) . Following this initial amplification cycle, denaturation and primer binding occur again, followed by further synthesis of complementary strands. The reaction therefore requires DNA molecules as a template, primers, nucleotides, and the enzyme DNA polymerase. Denaturation, primer hybridization, and elongation are controlled by temperature adjustment. The PCR process is thus typically performed in a thermocycler, usually with approximately 20 to 50 amplification cycles, the specific number of which is preset.

[0004] German patent application DE 10 2010 052 524 A1, for example, describes a PCR method for the qualitative and quantitative detection of nucleic acid sequences in real time, using a DNA probe labeled with a fluorophore. Under hybridization conditions, a mixture of duplexes is generated using primers, to which the labeled primer is bound. The labeled DNA probe is cleaved and quenched by the addition of a polymerase with exonuclease activity, thereby generating a measurable fluorescence signal.

[0005] US patent 8,119,352 B2 describes a multiplex amplification method for nucleic acids. In this method, the amplification of a first target molecule is analyzed; this amplification terminates as soon as a threshold is reached, and a second, new amplification is initiated at a different melting temperature.

[0006] US patent 2017 / 0106370 A1 discloses another amplification method for nucleic acids. In this method, the amplification of a first target molecule is analyzed, and this amplification is terminated upon reaching a predetermined amplification progress.

[0007] EP 3 101 144 A1 describes an analysis device for the amplification and sequencing of a polynucleotide.

[0008] An automated SELEX process is known from US 7,629,151 B2. In this process, all PCR reactions are monitored in real time. Each PCR reaction can be individually stopped by cooling. Disclosure of the invention Advantages of the invention

[0009] The present disclosure provides a method for carrying out a nucleic acid amplification process, wherein sample nucleic acids and reference nucleic acids are amplified in separate reaction mixtures. According to the invention, amplification signals are observed in real time, and the number of amplification cycles and / or the duration of the amplification process are dynamically adjusted depending on the amplification signals. For observing the amplification signals, the signals can be detected in a manner known per se, preferably using fluorescent probes to make the completed nucleic acid amplification detectable. The system can be configured such that the fluorescence increases proportionally with the amount of amplified products, and various fluorescent dyes can be used. For example, DNA dyes such as cyanine dyes (e.g., cyanide ...SYBR® (Green or PicoGreen®) or similar substances are used, which integrate into double-stranded DNA. Another possibility is the use of so-called FRET probes (Förster resonance energy transfer), in which a donor fluorochrome interacts with an acceptor fluorochrome. The detected and evaluated amplification signals are compared to the controls, and based on this, the number of amplification cycles and / or the duration of the amplification process are dynamically adjusted depending on the amplification signals. The core of the invention is therefore that the amplification signals of the sample and reference or control are detected and evaluated in real time or at multiple time points during the process, and predefined actions are executed based on this, primarily by dynamically adjusting the number of amplification cycles and / or the duration of the amplification process.

[0010] Preferably, this process is a real-time PCR, in which PCR cycles are performed to amplify sample nucleic acids and reference nucleic acids. In this preferred embodiment, the number of cycles is dynamically adjusted. Preferably, the amplification signals are related to each completed PCR cycle. Thus, for example, the signals can be detected and evaluated after each PCR cycle. Based on this virtually simultaneous PCR evaluation, a decision can be made after each cycle as to whether to start another cycle or stop the entire PCR process. For example, if a signal increase is detected in the sample containing the sample nucleic acid and / or in the sample containing the reference nucleic acid, the PCR can be stopped.Therefore, the PCR process time can be shortened by terminating the process after detecting the cycle threshold (CT value), which represents the beginning of the exponential rise of the amplification signal. Furthermore, this allows the PCR to be stopped at a point where a defined and known amount of PCR product has been generated. Thus, despite fluctuating PCR conditions, such as variations in the composition of the DNA-containing sample, a consistent amount of product is always produced during amplification.

[0011] Furthermore, the method according to the invention is also suitable for other amplification processes using DNA-synthesizing enzymes (amplification enzymes), for example for whole genome amplification. (whole genome amplification - WGA) or other, particularly isothermal, DNA amplification methods in which the amplification process essentially takes place at a constant temperature. These processes can employ, for example, various polymerases, helicases, ligases, or combinations of enzymes from the DNA replication ensemble. In these embodiments, the duration of the amplification process is dynamically adjusted depending on the amplification signals.

[0012] Real-time observation of amplification signals means that signals are not necessarily detected continuously, but rather that signals can be detected at specific, discrete time points, which can be assigned to individual PCR cycles, for example after each attachment step or each elongation step of a PCR cycle.

[0013] In this context, nucleic acid refers in particular to DNA, with DNA serving as a template ( template ) for amplification. Both the sample nucleic acids and the reference nucleic acids or control samples are carried in separate reaction mixtures. These reaction mixtures contain the respective nucleic acid as template DNA. They also contain the usual reagents for, for example, a PCR reaction, in particular primers that interact with the single strands of DNA at specific positions due to their complementary nucleotide sequences and define the starting point of DNA synthesis. Furthermore, a thermostable DNA polymerase and deoxyribonucleoside triphosphates, as building blocks for the DNA strand to be synthesized by the DNA polymerase, are included. In addition, the ions required for the function of the DNA polymerase and a suitable buffer solution are present.For other, especially isothermal amplification processes, for which the method can also be used to advantage, corresponding components, which are also known per se, are included in the reaction mixtures.

[0014] The procedure can be designed so that the observation and / or evaluation of the amplification signals in real time only starts after a predefined minimum number of amplification cycles and / or a predefined minimum duration of the amplification process has been completed. For example, this minimum number of cycles can be defined such that no signal is expected before this number of cycles or the minimum duration of the amplification has elapsed. This design has the advantage of saving resources for observing and evaluating the signals during phases in which no relevant results are expected. The minimum number of PCR cycles can, for example, be in the range of 10 or fewer. During these initial cycles, a baseline can be generated for subsequent analysis.

[0015] In a preferred embodiment of the method, the process is terminated when the signal strength of the amplification in the sample nucleic acid reaction reaches and / or exceeds the signal strength of the amplification in the reference nucleic acid reaction. In this case, it can be assumed that the amount of sample nucleic acid corresponds to the amount or concentration of the reference nucleic acid. This embodiment of the method allows, in particular, the determination of the initial concentration of the sample nucleic acid, after which the process can be terminated. Terminating the process before reaching a predefined maximum number of amplification cycles or before a predefined maximum process duration has the particular advantage of minimizing the formation of undesired byproducts, which can arise at the end of PCR reactions, especially with high cycle numbers (e.g., the formation of primer dimers).This facilitates further analysis during any subsequent characterization of the amplification products.

[0016] The amplification process can be terminated if a maximum number of amplification cycles and / or a predefined maximum process duration has been reached without a significant increase in the amplification signal being observed in the sample nucleic acid solution. This maximum number could, for example, be the number of PCR cycles chosen in conventional PCR experiments, such as 50 PCR cycles.

[0017] Overall, no new assay development is required for the method described here, as the usual reagents and reaction parameters for amplification processes are used. Only the process control, in particular the dynamic adjustment of the process duration and, for example, the number of PCR cycles and, if applicable, the composition of the controls, are placed in a new system context, depending on the application. The described method allows for the controlled, full automation of assay workflows without the need for intermediate quantification methods that would require sample collection followed by purification of the amplification products.

[0018] The procedure can be implemented, for example, by observing the amplification signals in relation to each amplification cycle performed. A significant increase in the signals classifies the respective cycle as an "amplification." A comparison of this classification result between samples with sample nucleic acids and with reference nucleic acids for the respective cycle is used for evaluation. Alternatively (or additionally) to individual amplification cycles, the signals can be related to definable time points during the process, with the signals being recorded at these defined time points. For example, the signals can be recorded at a rate between 1 second and 1 minute, meaning that signals are captured (e.g., by recording fluorescence images) at intervals of 1 second, 30 seconds, or 1 minute and evaluated, for example, as described above.Depending on the application, the observation time window can, for example, be between 1 second and 10 minutes, preferably between 30 seconds and 5 minutes. In a particularly preferred embodiment of the method, the results of the amplification process are evaluated as an indicator vector representation. For this purpose, amplification cycles or time points classified as "amplification" can, for example, be assigned the indicator value "1" and the other cycles or time points the indicator value "0".

[0019] This method allows for the particularly advantageous determination and / or control of the initial amount of the sample nucleic acids. For this purpose, preferably at least two reference samples with defined, i.e., known and predetermined, initial amounts of the reference nucleic acids are carried out in parallel. For example, one reference sample with a minimum initial amount or minimum initial concentration and at least one reference sample with a maximum initial amount or maximum initial concentration can be used. The largest initial amount (highest standard concentration) and the smallest or minimum initial amount (lowest standard concentration) allow for the establishment of a detection window.By using additional reference samples with concentrations within this range, several sub-intervals can be created, allowing for interval assignment to the initial concentration in the sample and enabling use for quality control, for example. The different concentrations of the reference or standard samples can differ by a factor of 10, for instance. As soon as amplification signals are detectable in the sample (indicator value "1"), the amplification process can be stopped, and the initial concentration or a concentration interval in the sample can be determined by comparing the respective indicator values ​​achieved up to that point with the standard concentrations. A particular advantage of this approach is that the time required to carry out the process can be reduced.It is not necessary to perform the maximum number of cycles or the maximum process duration required by conventional methods to detect amplification and its quantity. Instead, the process can be terminated after detecting the cycle threshold (CT value), which represents the beginning of the exponential rise of the amplification signal. The associated time savings are particularly significant when used in a [specific application / situation]. Point-of-Care (PoC) application is particularly advantageous.

[0020] In a further preferred embodiment of the method, it is used for infection detection. Here, at least one control sample containing a concentration of the nucleic acid to be detected (for example, a characteristic gene segment of a pathogen) is included, representing a lower detection limit. This detection limit can serve as the latest termination criterion for the amplification reaction. If a signal, particularly the "amplification" signal, is detected earlier in the sample containing the nucleic acids, the test can be considered positive. Additional control samples with varying concentrations of the nucleic acid to be detected can also be included, and in a valid test, the temporal sequence of amplification signals in the control samples should correspond to the sequence of concentrations.

[0021] In a further embodiment of the method, it is used for mutation detection. For this purpose, a reference sample with a defined concentration of the corresponding nucleic acid, containing 100% of the mutation to be detected, and preferably another reference sample with a defined concentration of the nucleic acid, containing 0% of the mutation to be detected (wild type), are used. Several mixture ratios of mutation nucleic acid and wild-type nucleic acid can be selected and used between these two limits.

[0022] According to the present invention, the method is used for whole genome amplification (WGA). A particular advantage here is that the amount of product generated during amplification can be controlled by carrying out corresponding comparison reactions with nucleic acid concentrations of known concentration. Especially in whole genome amplification, the problem of undesired byproducts can arise, particularly with high cycle numbers or after a longer amplification time, i.e., at the end of the WGA process. In contrast, the method described here offers the advantage that the process can be terminated as soon as a certain amount or concentration of product is reached, thus preventing the formation of undesired byproducts or minimizing their formation.

[0023] For the inventive use of the method for whole-genome amplification, at least one reference sample is provided, containing a defined concentration of the nucleic acid (DNA) of a reference genome. This first reference sample is preferably species-specific. For example, if a human genome is to be amplified, the DNA of another human being, or preferably a mixture from a large number of different humans, is used as the reference genome, so that genetic diversity can be taken into account. According to the invention, the defined concentration or quantity of the reference genome corresponds to a maximum usable quantity of DNA for whole-genome amplification systems. Furthermore, a second reference sample is preferably provided, which does not contain any nucleic acid to be amplified. template control Furthermore, a so-called quantity reference is preferably provided as a third control sample, containing a defined amount of nucleic acid from the reference genome, where this defined amount corresponds to the desired target amount of product in whole genome amplification. This third control sample does not contain any amplification enzyme. That is, no amplification takes place in this third control sample during the process.

[0024] By using fluorescent dyes that integrate into double-stranded DNA, and thus are independent of any amplification taking place, the fluorescent dye in this third reference sample is integrated into the already existing double-stranded DNA. The resulting fluorescence signal then corresponds to the signal that the process is intended to achieve in the actual sample for whole-genome amplification. The occurrence of amplification signals in the reference samples, compared to signals in the sample, defines various checkpoints that allow for controlled and automated execution of the process.

[0025] The described method is particularly well-suited for implementation in microfluidic systems, for example as a lab-on-a-chip system, where advantageously only very small sample volumes are required. The advantages of the described system are especially evident in connection with potential automation.

[0026] The various components for carrying out the described procedure can, for example, be provided as a kit for a user. This kit can contain, in particular, the reference samples, reagents, enzymes, and buffers required for the respective process.

[0027] The method can be implemented as a computer program designed to carry out the procedure. This computer program can be stored on a machine-readable data carrier and / or implemented in a suitable control unit for carrying out amplification processes.

[0028] Further features and advantages of the invention will become apparent from the following description of exemplary embodiments in conjunction with the drawings. The individual features can be implemented individually or in combination with one another.

[0029] The drawings show: Fig. 1 Schematic representation of the steps of a real-time PCR; Fig. 2 Illustration of the evaluation of fluorescence signals in a PCR process; Fig. 3 Illustration of the execution of an amplification process for determining the initial concentration of a sample; Fig. 4 Evaluation of an amplification process when performing an infection detection using an indicator vector representation; Fig. 5 Evaluation of an amplification process when performing a mutation detection using an indicator vector representation; Fig. 6 Illustration of the sequence of the inventive method in whole genome amplification; and Fig. 7 Schematic overview of the required device components for performing a real-time PCR according to the inventive method. Description of exemplary implementations

[0030] Fig. 1 Figure 10 schematically illustrates the process of real-time PCR according to a specific procedure. After the PCR process is started, the PCR cycles are initiated, with each step controlled by temperature regulation in a thermocycler. At regular intervals, particularly at defined time points within the PCR cycle (or analogously at specific time points in isothermal amplification processes), amplification signals are recorded and analyzed, for example, as fluorescence images. The choice of the appropriate time point may depend, for example, on the probe used. In the example shown here, a measurement is taken after each attachment step. In most cases, however, a measurement is taken after each elongation step. Each PCR cycle includes the denaturation step of the template DNA.Sample nucleic acids and reference nucleic acids are used as template DNA in separate reaction mixtures. After denaturation step 12, the attachment takes place (. annealing The respective primers are measured in step 13. In this example, the amplification signals are then measured in step 14. The measured signals are evaluated in step 15, specifically to determine whether the measured signal is classified as "amplification" or not. A comparison with the reference samples then determines whether further PCR cycles should be performed. If, for example, step 15 shows that the measured signal is background and not "amplification," the PCR cycle continues with the elongation step 16. The new PCR cycle then starts again with the denaturation step 12.However, if in step 15 it is determined that the measured signal is not to be considered a background signal but rather an "amplification", the PCR process can be terminated, and further analyses and evaluations of the PCR products formed may be carried out (step 17).

[0031] The detection of signals in step 14 is based on fluorescent probes that, in various known ways, such as incorporation during DNA synthesis or attachment or incorporation into DNA, make amplification detectable. A statistical test is then performed to determine whether this new data point can be classified as background data points already measured in previous PCR cycles, or whether the signal deviates significantly from the previously determined background and can be described as an "amplification."

[0032] It is advantageous to define minimum and maximum PCR cycle numbers as boundary conditions for the PCR process. The minimum number of cycles defines the earliest point at which a signal can be expected. These data points are automatically assigned to the background and are not tested for amplification. This minimum number of cycles can, for example, be set to 10 or less. During these initial cycles, a basic baseline can be generated. The maximum number of cycles can serve as a termination criterion if no amplification is detectable in the sample. This number is typically the same number of cycles specified in a classical PCR process.

[0033] Fig. 2 illustrates the evaluation (step 15 in Fig. 1 ) of the detected fluorescence signals. These fluorescence signals can be recorded in relation to individual PCR cycles or in relation to specific time points during the amplification process, particularly in the case of isothermal amplification processes (e.g., whole genome amplifications). Part A shows the background ( background - BG) or a baseline formed by individual data points (open circles) that are measured particularly in early PCR cycles and where amplification is not expected. The frame around the individual data points represents an estimated background with certain tolerances. This defined background is the basis for testing subsequent data points based on the measured fluorescence signals in subsequent PCR cycles or the subsequent amplification process. Subfigure B represents the subsequently measured data points as closed circles, based on further fluorescence signals in subsequent PCR cycles or the subsequent process, and which lie within the background frame. The most recent data point, represented by a cross, represents the current measurement, which is also within the background frame.It is assumed here that no amplification has taken place. Therefore, the old background is first calculated based on the data points from previous cycles; that is, the background is determined for all points except the current measurement (BG1). Once the current data point is available, a second background, BG2, is calculated, incorporating the current data point. Now, it can be statistically tested whether the two possible backgrounds, BG1 and BG2, differ significantly. The statistical evaluation can be performed according to the following specifications: Hypothesis H1: BG1 = BG2 Hypothesis H0: BG1 ≠ BG2 If, as in subfigure BP(H1) > P(H0), there is no significant difference and no amplification has taken place. The amplification process continues. However, if the background changes significantly due to the current data point (P(H1) < P(H0)), amplification can be assumed, as shown in subfigure C. This information forms the basis for the further course of action in the amplification process, and the process can be terminated.

[0034] Fig. 3 This illustrates the execution of the amplification process used to determine the initial concentration of sample DNA. This example is explained using a PCR process. This and the following examples can also be applied to isothermal amplification processes, for example, in which case the observed amplification signals are not assigned to individual PCR cycles but to discrete time points in the amplification process. Alongside sample 31, various reaction mixtures with standards 32, 33, 34, and 35 are carried out as reference samples. Here, standard 35 represents the highest standard concentration S1, and standard 32 the lowest standard concentration S4. In principle, any number of intermediate standard concentrations can be chosen between the maximum and minimum standard concentrations. In this example, there are two concentrations, S2 and S3.The number of different standard concentrations S1 to Sn determines the resolution of the concentration determination. All approaches are run in parallel after the start of the PCR process (step 30), and the amplification signals are recorded in step 36 during each PCR cycle. In step 37, it is evaluated whether amplification can be inferred or not. This can be done, in particular, using the method described in connection with [reference to relevant text]. Fig. 2 The numbers 1 and 0 shown in field 38 represent a classification as amplification ("1") or no amplification ("0"). The PCR process can be terminated if amplification is detected in sample 31. By comparing this with the amplification results of standard samples 32 to 35, it is then possible to determine the concentration range in which the initial concentration of DNA in sample 31 was found. If no amplification was detected in sample 31, but amplification occurred even at the lowest standard concentration 35, the PCR process can also be terminated, since the initial concentration in sample 31 is below the detection limit defined by the minimum standard concentration 35.This procedure is implemented by query 39, which checks whether amplification has been detected in either sample 31 or the reference sample 32 (or the standard with the lowest concentration). If so, the PCR process is terminated (step 40). If no amplification is detected in either sample 31 or the standard S4 with the lowest concentration (32), the next PCR cycle is executed in step 41. This method allows for the unambiguous assignment of a concentration interval. The concentration intervals are defined by the number of standards. It is expected that standards S1 to Sn, from highest to lowest concentration, will successively produce amplification signals as the PCR process progresses.If amplification is observed in sample 31 and simultaneously in standards S1 to S1i (i < n), then the initial concentration of sample 31 lies within the interval [S1i, S1i+1]. If the observation of amplification in the standards does not correspond to their concentration sequence, the test is not valid. Therefore, if a reaction with a lower standard concentration shows amplification in a PCR cycle where a standard with a higher concentration shows no amplification, the reactions are not equally efficient or comparable. The standard concentrations can be chosen, for example, to differ from each other by a factor of 10. This corresponds to quantification in the sense of classical real-time PCR.

[0035] Fig. 4 The procedure is illustrated using an indicator vector representation for infection detection. In addition to the actual sample 51, three standards 52, 53, and 54 are included. 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 maximum amount of DNA to be detected. Standard S1 represents the limit of detection. This limit of detection is the latest termination criterion for the reaction. If amplification is detected earlier in sample 51, and the order of amplification in the standards corresponds to their concentration order, the test is considered positive. Fig. 4 summarizes the evaluations of whether the reaction in a particular PCR cycle should be considered an amplification or not in an indicator vector ITogether, each reaction vessel or PCR reaction (samples and standards) has an entry in this vector, which is re-evaluated after each cycle. A reaction is considered an "amplification" if a signal is detectable above the background. This signal is recorded in the indicator vector. I the indicator value 1 ( true ). If no amplification is detected, the reaction indicator 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. The second standard, S2, which is quantitatively between the largest and smallest standards, follows next. The smallest standard, S1, which represents the detection limit, is in third position. The actual sample 51 is the last entry in the state vector. The experiment is initialized with I = [0,0,0,0]. In the Fig. 4 The four vectors shown represent a valid test. All twelve other possible cases are not permissible, and the test would have to be reported as invalid. In the case I If the signal is [1,1,1,0], it is near the detection limit. In this case, due to noise affecting reaction efficiency, one or more cycles may need to be added to ensure that any minor differences between individual reaction vessels do not lead to an error in the test decision. The last column of the graph shows the test result for each vector as positive (+) or negative (-). The reaction can be stopped as soon as either of these vectors is present.

[0036] Fig. 5 Figure 1 illustrates the procedure for applying mutation detection, also as an indicator vector representation. In mutation detection, a predefined amount of the sample DNA is typically introduced into the sample under investigation (61). Since the amount is predefined, the same amount of standard DNA or reference DNA is always used in standards 62, 63, and 64. Standard S1 (64) contains a template DNA in which 100% exhibits the mutation to be detected (M). This standard S1 forms the upper limit at which amplification should first be detected. The lower limit, and thus the final termination criterion of the reaction, is standard S3 (62), which contains 100% wild-type template (W). Several mixture ratios of mutation and wild-type DNA can be selected between these two limits. In this example, another standard S2 (63) is used, which contains 50% mutation (M = 50%).Adjusting the mixing ratios of mutational and wild-type DNA allows the sample to be divided into fraction bins, analogous to histograms. The standard chosen here, S2 with M = 50%, allows the mutation fraction to be categorized as more than 50% and less than 50%. With this approach, the ploidy of the gene can be determined, for example. Finer subdivisions are achieved by incorporating further standards and numerically estimating the efficiency. As in the previous example, using the... Fig. 4 The reaction is explained via the state vector. I The tests were checked and test decisions were made accordingly.

[0037] Fig. 6 Figure 1 illustrates the inventive method in connection with whole-genome amplification. In whole-genome amplification, all sequences present in a sample are amplified, not just defined DNA sequences addressed by primers. In classical whole-genome amplification, no fluorescent probes are used as is typical in real-time PCR; instead, the resulting amplification product is visualized and quantified using special dyes. These special dyes (e.g., PicoGreen®, SYBR® Green) bind to double-stranded DNA and fluoresce more intensely, so that an increase in fluorescence indicates successful amplification. Thus, if a fluorescence signal is detectable, this indicates the presence of double-stranded DNA, and therefore amplification can be interpreted as having occurred.This dye is added to the reaction mixtures in the process batches in a defined quantity. In addition to the actual sample 71 (S), three further reference samples 72, 73, and 74 are included in the whole-genome amplification process. Reference sample 72 does not contain template DNA as a so-called [missing information]. no template control (NTC). No amplification should be observed in this sample, as there is no DNA to be amplified. A further control sample 73, the DNA of a reference genome (RG), is included. This reference genome is expediently species-specific. For example, if a whole human genome is to be amplified, the genome of one or more other humans is used as the reference genome. The amount of the reference genome used corresponds, for example, to the maximum amount usable for a suitable whole genome amplification system. This reference genome in control sample 73 should therefore be the first to produce an amplification signal. After start 70, the reaction is carried out and tests for amplification are performed until the reference genome 73 and sample 71 are positive in the state vector (state 75). In state 75, both reference genome 73 and sample 71 show amplification, and the NTC control 72 shows no reaction.Amplification. Only in this state is sufficient DNA present in the sample, and up to this point, no non-specific primer amplification (formation of primer dimers) has occurred, as demonstrated by control 72. This state represents a first checkpoint. If the conditions for the first checkpoint are met, the reaction continues, but is now tested against a new termination criterion 76. The new test criterion 76 is the comparison of the amplification intensity in 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. Quantity reference 74 contains all components in the WGA reaction, as do the other reactions, with the exception of the amplification enzyme. Consequently, no amplification, i.e., no DNA synthesis, takes place in quantity reference 74 during the reaction.Only a reference fluorescence signal is provided by the introduced fluorescent dye. If the amplified sample 71 and the quantity reference 74 have the same fluorescence intensity, it can be assumed that essentially the same amount of double-stranded DNA is present in both samples, so that the reaction can be terminated in step 77. A further termination criterion could be that the NTC control 72 shows an amplification signal.

[0038] This method can also be applied to specific, targeted pre-amplification, where the amount of DNA synthesized during pre-amplification is controlled. Instead of the entire genome, specific primers are used, resulting in the appropriate up-copying of specific gene segments. Instead of a dye that binds to double-stranded DNA, a specific fluorescently labeled probe that generates a fluorescence signal dependent on the amount of synthesized DNA, such as a TaqMan® probe with a fluorophore and quencher, can be used. The quantity reference then includes the desired target amount of amplicon, an equivalent amount of cleaved probe (i.e., the same amount of free fluorophores and quencher), and a complementary residual amount of the probe.The underlying principle is that in a real-time PCR approach using a TaqMan® probe system, a defined initial quantity of probes (N0 = c0 V) is specified. When amplification begins, the probe is cleaved. The amount of probes and free fluorophores then depends on the resulting copy number NAmplicon. The remaining probes NS can be calculated using NS = N0 - NAmplicon. Instead of an NTC control, an additional reference is used as a termination criterion, which defines the limit of detection for amplification (LoA - ). limit of amplification ) makes it detectable. This involves using a minimal genome dilution. The first checkpoint is therefore the amplification time point at which an assay-specific, predefined genome dilution, i.e., the reference LoA, has been amplified.

[0039] The procedures of whole genome amplification according to the explanations at Fig. 6 and the mutation detection according to the explanations regarding Fig. 5 These steps can be linked together and designed as a controlled workflow for mutation detection. Microfluidic systems and / or pipetting robots can be used in this process. Such a fully automated workflow, particularly in conjunction with microfluidic systems, can offer a very advantageous application of the method according to the invention, for example in Point-of-Care -applications can be used.

[0040] Fig. 7Figure 1 illustrates the device components that can be used for the described real-time PCR processes. The basis for this is a device that enables optofluidic real-time PCR, thus allowing the readout of optical signals to be used to observe the amplification in the individual samples or PCR reactions based on fluorescence signals. Such a device includes a heating and cooling system 101 (thermocycler) that interacts with the various PCR reaction mixtures 102. Furthermore, the device has an optical unit 103 for reading out the amplification signals. In addition, a fluid handling system 104, such as a robotic system or a corresponding microfluidic system, may be included.Overall, it is advantageous to design such a system as a microfluidic system, since a microfluidic system can be operated with very small sample volumes and allows for partial or full automation. Furthermore, the system is equipped with a reaction control unit 105, which performs in-situ evaluation of the optical data. To realize the feedback-based real-time PCR system of the present invention, the reaction control unit 105 is designed to interact with all units of the system. In particular, the reaction control unit 105 can dynamically adjust the number of PCR cycles depending on the observed amplification signals.

Claims

1. Method for carrying out a process for amplification of nucleic acids (10), wherein sample nucleic acids (31; 51; 61; 71) and reference nucleic acids (32-35; 52-54; 62-64, 72-73) are amplified in separate reactions, wherein signals from the amplification are observed in real time and the number of amplification cycles and / or the duration of the amplification process are dynamically adjusted according to signals from the amplification, characterized in that the method is used for total genome amplification, wherein at least a first comparative sample (73) having a defined concentration of nucleic acid of a reference genome is run at the same time and the amount of the reference genome used corresponds to the maximum usable amount of DNA for total genome amplification systems.

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

3. Method according to Claim 1 or Claim 2, characterized in that the observation and / or evaluation of the signals from the amplification in real time starts when a specifiable minimum number of amplification cycles and / or a specifiable minimum duration of the amplification process has been carried out.

4. Method according to any of the preceding claims, characterized in that the process is ended when the signal strength of the amplification of the sample nucleic acids reaches and / or exceeds the signal strength of the amplification of the reference nucleic acids.

5. Method according to any of the preceding claims, characterized in that the process is terminated when a specifiable maximum number of amplification cycles and / or a specifiable maximum duration of the amplification process has been carried out.

6. Method according to any of the preceding claims, characterized in that the signals from the amplification are observed in relation to amplification cycles respectively carried out and / or in relation to definable time points and, when the signals rise significantly, the respective cycle or the respective time point is classified as "amplification" and a comparison of the classification between sample nucleic acids and reference nucleic acids is used for evaluation.

7. Method according to Claim 6, characterized in that the results of the process for amplification are evaluated as an indicator vector representation, wherein the cycles or time points classified as "amplification" are assigned to the indicator value "1" and the other cycles or time points are assigned to the indicator value "0".

8. Method according to any of the preceding claims, characterized in that the starting amount of the sample nucleic acids (31) is determined and / or checked by means of the method, wherein at least two comparative samples (32 - 35) having a defined starting amount of the reference nucleic acids are run in parallel at the same time.

9. Method according to any of the preceding claims, characterized in that the method is used as infection detection, wherein at least one comparative sample (52) having a concentration of the nucleic acid to be detected that represents a lower detection limit for the infection detection is run in parallel at the same time.

10. Method according to any of the preceding claims, characterized in that the method is used as mutation detection, wherein a comparative sample (64) having a defined concentration of nucleic acid containing a proportion of the mutation to be detected of 100% and a comparative sample (62) having a defined concentration of nucleic acid containing a proportion of the mutation to be detected of 0% are run at the same time.

11. Method according to any of the preceding claims, characterized in that a second comparative sample (72) without nucleic acid to be amplified and / or a third comparative sample (74) having a defined amount of nucleic acid of the reference genome are additionally run at the same time, wherein the defined amount of the third comparative sample corresponds to the desired target amount of amplification product in the total genome amplification, and wherein the reaction containing the third comparative sample does not contain an amplification enzyme, and wherein the signal from the amplification is based on the use of fluorescent dyes which are intercalated into double-stranded DNA.