Abstract
A method for carrying out a process for an amplification of nucleic acids with sample nucleic acids and reference nucleic acids being amplified in separate reaction batches. Signals of the amplification are observed in real time. A number of amplification cycles and/or a duration of the amplification process are dynamically adjusted depending on the observed signals of the amplification.
Claims
1. A method for performing a process for amplification of nucleic acids, comprising: amplifying sample nucleic acids and reference nucleic acids in separate reaction preparations; detecting signals of the amplification in real-time; and dynamically adjusting a number of amplification cycles and/or a duration of an amplification process based on the detected signals of the amplification.
2. The method as claimed in claim 1, wherein: the amplification of the sample and the reference nucleic acids is performed in a context of a real-time polymerase chain reaction, and the amplification cycles are PCR cycles.
3. The method as claimed in claim 1, wherein: the detecting, observing, and/or evaluation of the signals of the amplification in real-time starts when a specifiable minimum number of the amplification cycles and/or a specifiable minimum duration of the amplification process has been performed.
4. The method as claimed in claim 1, further comprising: ending the process when a detected signal intensity of the amplification of the sample nucleic acids reaches and/or exceeds a detected signal intensity of the amplification of the reference nucleic acids.
5. The method as claimed in claim 1, further comprising: terminating the process when a specifiable maximum number of the amplification cycles and/or a specifiable maximum duration of the amplification process has been performed.
6. The method as claimed in claim 1, wherein: the signals of the amplification are detected in relation to respectively performed amplification cycles and/or in relation to definable time points and classified as “amplification” in an event of a significant rise in the signals of the respective cycle or the respective time point and a comparison of the classification between sample nucleic acids and reference nucleic acids is used for an evaluation.
7. The method as claimed in claim 6, further comprising: evaluating results of the process for amplification as an indicator vector display, wherein the amplification cycles or time points classified as “amplification” are assigned to the indicator value “1” and the other amplification cycles or time points are assigned to the indicator value “0”.
8. The method as claimed in claim 1, wherein: a starting amount of the sample nucleic acids is ascertained and/or checked, and at least two comparative samples having a defined starting amount of the reference nucleic acids are concomitantly run in parallel.
9. The method as claimed in claim 1, further comprising: detecting an infection detection based on the amplifying, wherein at least one comparative sample having a concentration of the nucleic acid to be detected that represents a lower detection limit for the infection detection is concomitantly run in parallel.
10. The method as claimed in claim 1, further comprising: detecting a mutation based on the amplification, wherein a comparative sample having a defined concentration of nucleic acid having a 100% proportion of the mutation to be detected and a comparative sample having a defined concentration of nucleic acid which contains a 0% proportion of the mutation to be detected are concomitantly run.
11. The method as claimed in claim 1, wherein the amplifying comprises: amplifying a whole genome, wherein at least one first comparative sample having a defined concentration of nucleic acid of a reference genome is concomitantly run.
12. The method as claimed in claim 11, wherein: a second comparative sample without nucleic acid to be amplified and/or a third comparative sample having a defined amount of nucleic acid of the reference genome are additionally concomitantly run, the defined amount of the third comparative sample corresponds to the desired target amount of amplification product in the amplification of the whole genome, the reaction preparation of the third comparative sample contains no amplification enzyme, and the detected signal of the amplification is based on a use of fluorescent dyes which intercalate into double-stranded DNA.
13. The method as claimed in claim 11, wherein the method is used for a check of a preamplification.
14. The method as claimed in claim 1, wherein: the process for amplification is a nested PCR comprising a first multiplex PCR and at least one second singleplex PCR, and the amount of the nucleic acid amplified in the first multiple PCR is checked.
15. The method as claimed in claim 1, wherein a computer program is configured for performance of the method.
Description
[0024] In the drawings:
[0025] FIG. 1 shows a schematic representation of the steps of a real-time PCR with implementation of the method according to the invention;
[0026] FIG. 2 show an illustration of the evaluation of fluorescent signals in a PCR process in the context of the method according to the invention;
[0027] FIG. 3 show an illustration of the performance of an amplification process as per the method according to the invention for determination of the starting concentration of a sample;
[0028] FIG. 4 shows an evaluation of an amplification process as per the method according to the invention in the case of performance of an infection detection on the basis of an indicator vector display;
[0029] FIG. 5 shows an evaluation of an amplification process as per the method according to the invention in the case of performance of a mutation detection on the basis of an indicator vector display;
[0030] FIG. 6 show an illustration of the course of the method according to the invention in the case of a whole genome amplification and
[0031] FIG. 7 shows a schematic overview of the necessary instrument components for performance of a real-time PCR as per the method according to the invention.
DESCRIPTION OF THE INVENTION
[0032] FIG. 1 schematically shows the course of a real-time PCR as per the method according to the invention. After the start 11 of the PCR process, the PCR cycles are started, these individual steps being carried out by a control of the temperature in a thermocycler. At regular intervals, especially at defined time points within the PCR cycle (or analogously at particular time points in isothermal amplification processes), signals of the amplification are captured and, for example, recorded and evaluated as fluorescent images. The choice of the respective suitable time point can, for example, depend on the probe respectively used. In the example shown here, measurement is, for example, carried out after each attachment step. However, in most cases, measurement is carried out after each elongation step. Each PCR cycle comprises the step of denaturation 12 of the template DNA.
[0033] The template DNA used are sample nucleic acids and reference nucleic acids in separate reaction preparations. The denaturation step 12 is followed by the attachment (annealing) of the respective primers in step 13. In this example, this is followed by the measurement of the signals of the amplification in step 14. The measured signals are evaluated in step 15, a check in particular being made to determine whether the measured signal is classified as “amplification” or not. Especially in comparison with the reference samples, a decision is then made as to whether further PCR cycles are performed or not. For example, if it is established in step 15 that the measured signal should be classified as background, i.e., not as “amplification”, the PCR cycle is continued with the elongation step 16. Thereafter, the new PCR cycle starts with the denaturation step 12. However, if it is established in step 15 that the measured signal should not be rated as background signal, but should be classified as “amplification”, the PCR process can be terminated, and optionally further analyses and evaluations of the PCR products formed can be carried out (step 17).
[0034] The detection of the signals in step 14 is based on fluorescent probes, by means of which an amplification which has taken place is made detectable in various ways known per se, for example by incorporation in the DNA synthesis or by attachment or intercalation into the DNA. Especially statistical testing is then carried out to determine whether this new data point can be classified as background with data points already measured in previous PCR cycles or whether the signal significantly deviates from the hitherto determined background signal and can be referred to as “amplification”.
[0035] Expediently, a minimum and a maximum PCR cycle number are specified as boundary conditions for the PCR process. The minimum cycle number defines from when a signal can be expected at the earliest. These data points are automatically assigned to the background and are not tested for amplification. Said minimum cycle number can, for example, be set to 10 or smaller. During these initial cycles, a base line can be generated. The maximum cycle number can form a termination criterion for the case of no amplification being detectable in the sample. Said number is typically the number of cycles that is also specified in a classic PCR process.
[0036] FIG. 2 illustrates the evaluation (step 15 in FIG. 1) of the detected fluorescent signals. Said fluorescent signals can be captured in relation to individual PCR cycles or else in relation to particular time points during the amplification process, especially in the cases of isothermal amplification processes (e.g., in the case of whole genome amplifications). Subfigure A shows the background (BG) or a baseline that is formed by individual data points (open circles) which are measured especially in early PCR cycles and in which there can be no assumption of an amplification. The frame around the individual data points represents an estimated background with certain tolerances. This thus defined background is the basis of the tests of the subsequent data points on the basis of the measured fluorescent signals in following PCR cycles or in the following amplification process. Subfigure B depicts the subsequently measured data points as closed circles, which are based on further fluorescent signals in subsequent PCR cycles or in the subsequent process and which are located within the frame of the background. The most recent data point depicted with a cross represents the current measurement value, which is likewise located within the frame of the background. Here, it can be assumed that no amplification has taken place. What is thus initially calculated on the basis of the data points from preceding cycles is the old background, i.e., the background is ascertained for all points with the exception of the current measurement value (BG1). When the current data point is available, a second background BG2 is calculated, the current data point being included. It is then possible to carry out statistical testing to determine whether the two possible backgrounds BG1 and BG2 significantly differ. The statistical evaluation can be done as per the following specification:
Hypothesis H1: BG1=BG2
Hypothesis H0: BG1≠BG2
[0037] If, as in subfigure B, P(H1)>P(H0), there is no significant difference and no amplification has taken place. The amplification process is continued. By contrast, if the background changes significantly owing to the current data point (P(H1)<P(H0)), an amplification can be assumed, as depicted in subfigure C. This information is the basis of further action in the amplification process and the process can be ended.
[0038] FIG. 3 illustrates the embodiment of the amplification process with which a starting concentration of a sample DNA is determined. This example is elucidated with reference to a PCR process. This example and the following examples can also, for example, be applied to isothermal amplification processes, wherein the observed amplification signals are then assigned not to individual PCR cycles, but to discrete time points in the amplification process. Concomitantly run in parallel with the sample 31 are various reaction preparations containing standards 32, 33, 34 and 35 as comparative samples. Here, the standard 35 represents the largest standard concentration S.sub.1 and the standard 32 represents the smallest standard concentration S.sub.4. Between the maximum and the minimum standard concentration, as many intermediate stages of the standard concentrations as desired can be chosen in principle. In this example, there are two concentrations S.sub.2 and S.sub.3. The number of different standard concentrations S.sub.1 to S.sub.n determines the resolution of concentration determination. All the preparations are run in parallel after the start 30 of the PCR process and, during the individual PCR cycles, the signals of the amplification are captured in step 36. In step 37, what is evaluated is whether an amplification can be deduced or not. This can be done especially by means of the method as described in connection with FIG. 2. The numerals 1 and 0 depicted in the field 38 stand for a classification as amplification (“1”) or no amplification (“0”). The PCR process can be terminated when an amplification is established for the sample 31. In comparison with the amplification results for the standard samples 32 to 35, it is then possible to deduce the concentration interval in which the starting concentration of the DNA in the sample 31 was present. If no amplification could be established for the sample 31, but an amplification already appeared for the lowest standard concentration 35, the PCR process can likewise be terminated, since the starting concentration in the sample 31 is below the detection limit which is defined by the minimum standard concentration 35. This approach is realized by the query 39, by a check being made between the sample 31 and the comparative sample 32 or the standard having the lowest concentration to determine whether an amplification was established for one of the two preparations. In this case, the PCR process is ended (step 40). If an amplification cannot be established either for the sample 31 or for the standard S4 having the lowest concentration 32, the next PCR cycle is carried out in step 41. This method allows an unambiguous assignment of a concentration interval. The concentration intervals are, then, defined by the number of standards. What is to be expected here is that the standards S.sub.1 to S.sub.n provide amplification signals successively from the greatest concentration up to the lowest concentration as the PCR process advances. If an amplification is established for the sample 31, and at the same time an amplification for the standards S.sub.1 to S.sub.i (i<n), the starting concentration for the sample 31 lies in the interval [S.sub.i, S.sub.i+1]. If the establishment of an amplification for the standards is not in agreement with the order of their concentrations, the test is not valid. Thus, if one preparation having a lower standard concentration shows an amplification at a PCR cycle at which a standard having a higher concentration does not yet show any amplification, the reactions are not equally efficient or not comparable. The choice of the standard concentrations can, for example, be made such that they each differ from one another by a factor of 10. This corresponds to a quantification in the context of a classic real-time PCR.
[0039] FIG. 4 illustrates the method by means of an indicator vector display for an infection detection. In addition to the actual sample 51, three standards 52, 53, 54 are concomitantly run, wherein the standard 52 is the standard S.sub.1 having the lowest concentration of the DNA to be detected, the standard 53 is the standard S.sub.2 having a medium concentration of the DNA to be detected and the standard 54 is the standard S.sub.3 having a maximum amount of the DNA to be detected. The standard S.sub.1 represents the detection limit. Said detection limit is the latest termination criterion of the reaction. If an amplification is established earlier for the sample 51 and if the order of the appearance of the amplification for the standards corresponds to the order of their concentration, the test is rated as positive. FIG. 4 summarizes, in an indicator vector I, the evaluations to determine whether the reaction can be rated as amplification or not at a particular PCR cycle. In said vector, each reaction vessel or each PCR preparation (samples and standards) has an entry which is re-evaluated after each cycle. A reaction is rated as “amplification” if a signal is detectable above the background. The indicator value 1 (true) is assigned thereto in the indicator vector I. If no amplification is establishable, the indicator of the reaction is set to 0 (false). In this example, the standard S.sub.3 is the largest standard and is listed on the left as upper detection limit. The second standard S.sub.2, which in terms of amount is between the largest and the smallest standard, follows next. Following at the third position is the smallest standard S.sub.1, which represents the detection limit. Following as the last entry in the state vector is the actual sample 51. The experiment is initialized with I=[0, 0, 0, 0]. FIG. 4 shows the four vectors which 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 of I=[1, 1, 1, 0], the signal is in the range of the detection limit. In this case, one or more cycles can optionally be attached owing to noise of reaction efficiency, so that any small differences present between the individual reaction vessels do not lead to an error in the test decision. In the last column of the display, the test result is displayed as positive (+) or as negative (−) for the respective vectors. Once one of these vectors is present, the reaction can be terminated.
[0040] FIG. 5 illustrates the embodiment of the method for application of a mutation detection, likewise as an indicator vector display. In the case of a mutation detection, what is generally used is a predefined amount of the sample DNA in the sample 61 to be tested. Since the amount is predefined, the same amount of standard DNA or reference DNA is always used in the standards 62, 63 and 64. The standard S.sub.1 64 contains an initially charged template DNA in which 100% has the mutation (M) to be detected. Said standard S.sub.1 forms the upper limit at which an amplification should be detected first. The lower limit and hence the last termination criterion of the reaction is a standard S.sub.3 62 which contains 100% wild-type template (W). Between these two limits, it is possible, then, to choose multiple mixture ratios of mutation DNA and wild-type DNA. In this example, a further standard S.sub.2 63 is provided that contains 50% mutation (M=50%). The setting of mixture ratios of mutation DNA and wild-type DNA allows the division of the sample into proportion bins, analogous to histograms. The standard S.sub.2 with M=50% that is chosen here allows a categorization of the proportion of mutation of greater than 50% and less than 50%. In this approach, it is thus, for example, possible to determine the ploidity of the gene. Finer subdivisions are achieved by the insertion of further standards and by a numerical estimation of the efficiency. As elucidated in the previous example with reference to FIG. 4, the reaction is checked via the state vector I and test decisions are accordingly made.
[0041] FIG. 6 illustrates the method in connection with a whole genome amplification. In the case of a whole genome amplification, all sequences which occur in a sample are amplified, i.e., not just defined DNA sequences which are addressed via primers. In a classic whole genome amplification, fluorescent probes, as is customary in a real-time PCR, are not used; instead, the amplification product which forms is visualized and quantified by the use of specific dyes. Said specific dyes (e.g., PicoGreen®, SYBR® Green) intercalate into double-stranded DNA and, in doing so, emit light more intensely, meaning that a rise in fluorescence indicates an amplification that has taken place. Thus, if a fluorescent signal is detectable, this indicates the presence of double-stranded DNA, and so this can thus be rated as amplification. Said dye is added in a defined amount to the reaction mixtures in the preparations for the process. Besides the actual sample (S), three further comparative samples 72, 73 and 74 are concomitantly run in the process for the whole genome amplification. The comparative sample 72 contains no template DNA as so-called no template control (NTC). In the case of said sample, no amplification should be establishable, since DNA to be amplified is not present. As further comparative sample 73, the DNA of a reference genome (RG) is concomitantly run. Said reference genome is expediently species-specific. For example, if a human genome is to be amplified as a whole, the genome of one or more other persons is used as reference genome. The amount used of the reference genome corresponds, for example, to the maximum usable amount for a suitable whole genome amplification system. Said reference genome in the comparative sample 73 should therefore provide an amplification signal first of all. After the start 70, the reaction is performed and is tested for amplification until the reference genome 73 and the sample 71 are positive in the state vector (state 75). Thus, in the state 75, both reference genome 73 and sample 71 show an amplification and the NTC control 72 shows no reaction or amplification. It is only in this state that sufficient DNA is present in the sample, and up to this state, no nonspecific primer amplification (formation of primer dimers) has taken place, as has been shown on the basis of the control 72. This state represents a first checkpoint. If the conditions for the first checkpoint have been met, the reaction is continued, but now tested for a new termination criterion 76. The new test criterion 76 is the comparison of the intensity of the amplification for the sample 71 and the quantitative reference 74. Said quantitative reference 74 contains the desired amount of reference genome, this corresponding to the desired amount of product in the whole genome amplification. Here, the quantitative reference 74 contains all the components in the WGA preparation, like the other preparations, with the exception of the amplification enzyme. As a result, no amplification, i.e., no DNA synthesis, takes place in the quantitative reference 74 during the reaction. What is provided is only a reference fluorescent signal due to the initially charged fluorescent dye. If, then, amplified sample 71 and the quantitative reference 74 have the same fluorescence intensity, it can be assumed that the same amount of double-stranded DNA is present in principle in both preparations, and so the reaction can be terminated in step 77. As a further termination criterion, what can be provided is that the NTC control 72 shows an amplification signal.
[0042] This method can also be applied to a specific, targeted preamplification in which the amount of DNA that is synthesized in a preamplification is checked. In this case, specific primers are used instead of the whole genome, the result being that specific gene segments are accordingly highly copied. What can also be used here as probe instead of a dye which intercalates at double-stranded DNA is a specific fluorescently labeled probe which generates a fluorescent signal depending on synthesized DNA, for example a TaqMan® probe with fluorophore and quencher. The quantitative reference then contains the desired target amount of amplified material, an equivalent amount of cleaved probe, i.e., the same amount of free fluorophores and quenchers, and a complementary residual amount of the probe. The basis of this is that, in the case of a real-time PCR preparation in a TagMan® probe system, a defined starting amount of the probes (N.sub.0=c.sub.0V) is specified. When the amplification starts, the probe is cleaved. The amount of probes and free fluorophores is then dependent on the copy number N.sub.Amplicon that arises. The residual probes N.sub.S can be calculated using N.sub.S=N.sub.0−N.sub.Amplicon. Instead of an NTC control, what is concomitantly run as termination criterion is a further reference which makes a detection limit for the amplification (LoA—limit of amplification) detectable. Here, a minimum genome dilution to be used is used. Here, the first checkpoint is thus the amplification time point at which an assay-specific, predefined genome dilution, i.e., the reference LoA, was amplified.
[0043] The methods of the whole genome amplification as per the explanations in relation to FIG. 6 and the mutation detection as per the explanations in relation to FIG. 5 can be linked to one another and be configured as a monitored workflow for a mutation detection. Here, microfluidic systems and/or pipetting robots can be used. Such a fully automatic workflow can, especially in connection with microfluidic systems, offer a very advantageous possible use of the method according to the invention which can, for example, be used in point-of-care applications.
[0044] FIG. 7 illustrates the instrument components which can be used for the described real-time PCR processes. Here, the basis is formed by an instrument which makes an optofluidic real-time PCR possible and thus allows a signal readout of optical signals in order to be able to observe the amplification in the individual samples or PCR reactions on the basis of fluorescence signals. Such an instrument comprises a heating and cooling system 101 (thermocycler) which interacts with the various PCR reaction preparations 102. Furthermore, the instrument has an optical unit 103 which effects the readout of the amplification signals. Furthermore, a device for fluid handling 104 can be provided, for example a robot system or a corresponding microfluidic system. Altogether, it is advantageous to configure such a system as a microfluidic system, since a microfluidic system can be operated with very small sample volumes and allows semiautomation or full automation. The system is furthermore provided with a reaction control unit 105 which effects an in situ evaluation of the optical data. To realize the feedback real-time PCR system of the present invention, the reaction control unit 105 is configured such that it can interact with all units of the system. The reaction control unit 105 can especially control the dynamic adjustment of the number of PCR cycles depending on the observed signals of the amplification.
