Method and Device for Carrying Out a qPCR Method

Abstract

The disclosure relates to a method for operating a quantitative polymerase chain reaction (qPCR) method, having the following steps: cyclically carrying out qPCR cycles; measuring the fluorescence in each qPCR cycle in order to obtain a qPCR curve of intensity values; determining the reaction efficiency (η) for each cycle; correcting the intensity value of each cycle on the basis of the reaction efficiency (η) determined for the cycle in question in order to obtain a corrected qPCR curve; and operating the qPCR method on the basis of the corrected qPCR curve.

Claims

1. A method for conducting a quantitative polymerase chain reaction (qPCR) process the method comprising: cyclically executing qPCR cycles; measuring a fluorescence at each qPCR cycle to obtain a qPCR curve composed of intensity values; determining a reaction efficiency for each qPCR cycle; correcting a respective intensity value of each respective qPCR cycle depending on the reaction efficiency determined for the respective qPCR cycle to obtain a corrected qPCR curve; and conducting the qPCR process depending on a shape of the corrected qPCR curve.

2. The method as claimed in claim 1 further comprising: conducting a reaction liquid into a reaction chamber, wherein the reaction efficiency is determined depending on an area of at least one bubble in the reaction chamber.

3. The method as claimed in claim 2 further comprising: recording an image of the reaction chamber with a camera; and determining the area of the at least one bubble with pattern recognition methods applied to the image of the reaction chamber.

4. The method as claimed in claim 3, the determining further comprising: determining the reaction efficiency with a brightness of at least one pixel of the image that corresponds to the reaction liquid and the area of the at least one bubble as a proportion of a total area of the reaction chamber.

5. The method as claimed in claim 1, the determining further comprising: determining the reaction efficiency depending on an area of at least one bubble in a reaction chamber for at least two of (i) a denaturation process, (ii) an annealing and (iii) a elongation process.

6. The method as claimed in claim 1 further comprising: determining, using the corrected qPCR curve, based on a classification method, whether a DNA strand segment to be detected is present.

7. The method as claimed in claim 1, the conducting the qPCR process further comprising: signaling that a ct value is determinable; and determining the ct value from a parameterized presence function in response to a presence of the DNA strand segment to be detected being established.

8. A device for conducting a quantitative polymerase chain reaction (qPCR) process, the device being configured to: cyclically execute qPCR cycles; measure a fluorescence at each qPCR cycle to obtain a qPCR curve composed of intensity values; determine a reaction efficiency for each qPCR cycle; correct a respective intensity value of each respective qPCR cycle depending on the reaction efficiency determined for the respective qPCR cycle to obtain a corrected qPCR curve; and conduct the qPCR process depending on a shape of the corrected qPCR curve.

9. The method as claimed in claim 1, wherein the method is carried out by executing a computer program.

10. A non-transitory electronic storage medium storing a computer program for conducting a quantitative polymerase chain reaction (qPCR) process, the computer program being configured to, when executed by a computer, cause the computer to: cyclically execute of qPCR cycles; measure a fluorescence at each qPCR cycle to obtain a qPCR curve composed of intensity values; determine a reaction efficiency for each qPCR cycle; correct a respective intensity value of each respective qPCR cycle depending on the reaction efficiency determined for the respective qPCR cycle to obtain a corrected qPCR curve; and conduct the qPCR process depending on a shape of the corrected qPCR curve.

11. The method as claimed in claim 2, the conducting the reaction liquid further comprising: conducting a reaction liquid into a reaction chamber in each qPCR cycle.

12. The method as claimed in claim 2, wherein the reaction efficiency is determined depending on an area of at least one bubble in the reaction chamber for an elongation process.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Embodiments will be more particularly elucidated below on the basis of the accompanying drawings, where:

[0035] FIG. 1 shows a schematic depiction of a cycle of a PCR method;

[0036] FIG. 2 shows a system for carrying out a PCR method;

[0037] FIG. 3 shows a schematic depiction of a typical qPCR curve comprising a plot of intensity values;

[0038] FIG. 4 shows a measured plot of a qPCR curve;

[0039] FIGS. 5a and 5b show ideal plots of the qPCR curve in the case of a nondetectable substance and a detectable substance, respectively; and

[0040] FIG. 6 shows a flowchart to illustrate a method for conducting a qPCR measurement;

[0041] FIG. 7 shows a photographic image of a reaction chamber containing a bubble;

[0042] FIG. 8 shows a flowchart to illustrate a further method for conducting a qPCR measurement.

DESCRIPTION OF EMBODIMENTS

[0043] FIG. 1 shows a schematic depiction of a PCR method known per se, comprising the steps of denaturation, annealing and elongation.

[0044] In the annealing step S1, the double-stranded DNA in a substance is broken up into two individual strands at a high temperature of, for example, above 90° C. In a subsequent annealing step S2, a so-called primer is bound to the individual strands at a particular DNA position marking the start of a DNA strand segment to be detected. Said primer represents the starting point of an amplification of the DNA strand segment. In an elongation step S3, the complementary DNA strand segment is synthesized on the individual strands from free nucleotides added to the substance, starting at the position marked by the primer, with the result that the previously split individual strands have been completed to form complete double strands at the end of the elongation step.

[0045] By providing the free nucleotides or the primer with fluorescent molecules which exhibit fluorescence properties only when bound to the DNA strand segment, it is possible, by determining an intensity of a fluorescence following the elongation step S3, to obtain an intensity value through an appropriate measurement. What is assigned to the measured intensity of the fluorescent light is an intensity value.

[0046] The method comprising steps S1 to S3 is executed cyclically and the intensity values are recorded in order to obtain a plot of intensity values as a qPCR curve.

[0047] FIG. 2 depicts a system 10 for carrying out a PCR method. The system comprises three reaction chambers—the denaturation chamber 11, the annealing chamber 12 and the elongation chamber 13 for carrying out denaturation, annealing and elongation, respectively—each of which is connected to an optical system for measuring an intensity value. The optical system comprises a respective camera 14, 15, 16, which cameras are connected to a control unit 20 in which the camera images are evaluated. To this end, the reaction chambers 11, 12, 13 can be closed on at least one side with a transparent face, to which the respective camera 14, 15, 16 is directed. The cameras are used to capture a camera image of the respective reaction chamber and to provide said camera image to the control unit 20. The cameras 14, 15, 16 are suitable for detecting a fluorescent light of the PCR method. The control unit 20 is designed to carry out image processing of the recorded camera images and to determine an intensity value therefrom according to one of the methods described below.

[0048] The plot of intensity values ideally has the shape depicted in FIG. 3. FIG. 3 shows a plot of normalized intensity against the cycle index z. Said plot is divided into three sections, namely a baseline section B, in which the fluorescence of the incorporated fluorescent molecules is still indistinguishable from a background fluorescence, an exponential section E, in which the intensity values are visible and rise exponentially, and in a plateau section P, in which there is flattening of the rise in intensity values, since the reagents used (solution containing nucleotides) have been consumed and no further binding to broken-up individual strands is taking place.

[0049] FIG. 4 depicts, by way of example, a plot of the intensity values obtained in a real measurement as a qPCR curve. Strong fluctuations are evident, and these may result from background fluorescence, thermal noise, fluctuations in the reagent concentrations, and bubbles and artifacts in the fluorescence volume. It is evident that it is not readily possible to determine the baseline section, exponential section and plateau section of the qPCR curve.

[0050] FIGS. 5a and 5b show ideal plots of a qPCR curve without the presence of a DNA strand segment to be detected and with the presence of a DNA strand segment to be detected, respectively.

[0051] FIG. 6 depicts a flowchart to illustrate a method which is executed in the control unit. The method can be implemented in a data processing device as hardware and/or software.

[0052] In step S11, a PCR measurement method is started.

[0053] In step S12, the steps of denaturation, annealing and elongation are carried out as described above and an intensity value is determined in each cycle at the end of the elongation step. This is done by taking images of the elongation chamber 13 with the aid of the camera 16. The intensity value can be determined by taking into account bubble formation in the reaction chamber for elongation. FIG. 7 shows, by way of example, a photographic image of a reaction chamber containing an air bubble in the reaction liquid.

[0054] Methods known per se can be used to determine the presence of a bubble in the reaction chamber. Such methods, what can be performed are thresholding (Otsu's method), edge detection, Hough transform, and detection with the aid of data-based machine learning methods, for example with use of deep neural networks and the like. From the detected bubble, it is possible to determine the bubble area in the image, i.e., the area occupied by the bubble and the halo of the bubble. From the ratio between the bubble area and the total area of the reaction chamber, it is possible to determine a bubble volume quotient which specifies the volume fraction in the reaction chamber that is occupied by the bubble and hence displaces a corresponding portion of the reaction liquid. A measured intensity value is therefore only yielded by the remaining reaction liquid and can be reduced by the factor 1−V.sub.b according to the volume V.sub.b of the bubble, since the brightness of the fluorescence in the reaction chamber is only caused by the remaining reaction liquid.

[0055] An alternative approach can consist in selecting only the brightness of one or more pixels in the image of the reaction chamber and disregarding pixels which are part of a bubble or an associated halo. The brightnesses of pixels which can be assigned to the reaction liquid can be averaged in order to obtain a corresponding intensity value for creation of the qPCR curve. Pixels relevant to averaging can be selected by using classification methods, especially with use of machine learning methods. Said machine learning methods can be used to carry out so-called semantic segmentation. In semantic segmentation, one of a plurality of classes is assigned to each pixel of a camera image.

[0056] For this purpose, it is possible to use a data-based method comprising a classification model which has been trained with pixelwise labeled data. For this specific application, multiple n-ary classifications are conceivable: [0057] 1. Binary classification: The class “part of a bubble” or “not part of a bubble” is assigned to each pixel. Grayscale value determination is then only done via the pixels of the class “not part of a bubble” that lie within the reaction volume. This method therefore requires that the position, size and orientation of the reaction chamber be known and be unalterable relative to the camera. [0058] 2. Ternary classification: This variant adds the additional class “not part of the reaction volume” to variant 1. As a result, this method no longer requires that the position, size and orientation of the reaction chamber be known and be unalterable relative to the camera. [0059] 3. Ternary classification, second variant: This variant adds the additional class “part of the halo of a bubble” to variant 1. It may prove to be useful to evaluate the pixels in the halo of a bubble separately, since the halo can also illuminate part of the actual reaction volume. This method therefore also requires that the position, size and orientation of the reaction chamber be known and be unalterable relative to the camera. [0060] 4. Quaternary classification: This variant corresponds to a combination of variants 2 and 3. Thus, with said variant, it is possible not only to separately evaluate pixels in halos, but also to establish and compensate for changes in the relative orientation of the reaction chamber to the camera. [0061] 5. Binary classification. With this variant, the class “part of the edge of a bubble” or “not part of the edge of a bubble” is assigned to each pixel. This corresponds to edge detection. Grayscale value determination is then only done via the pixels that are completely surrounded by pixels of the class “part of the edge of a bubble”. This therefore requires that the position, size and orientation of the reaction chamber be known and be unalterable relative to the camera.

[0062] The intensity values thus obtained can be corrected in step S13 by taking reaction efficiency into account. In the prior art, the reaction efficiency is assumed to be constant, especially to be 1 in the ideal scenario. However, for actual systems, it can be assumed that the reaction efficiency varies in each cycle, meaning that the intensity values and the qPCR curve determined therefrom are incorrect.

[0063] It is assumed herein that the reaction mixture displaced by bubbles in the reaction chamber cannot contribute to amplification, since it is present in channels or other chambers, but not in the reaction chamber. The reaction efficiency therefore becomes worse.

[0064] The number of DNA strand segments per cycle under a constant reaction volume is defined as follows:

n.sub.i+1=n.sub.i(1+η)

[0065] where n.sub.i corresponds to the number of DNA strand segments in cycle i and η corresponds to the chemical reaction efficiency between 0 and 1. In the simplest case, η is assumed to be 1. In a particular embodiment, this factor can also be determined by a series of experiments and thus more closely approximated.

[0066] If the displaced bubble volume V.sub.B,i is then specified as the bubble volume quotient, as the quotient between the area occupied by the bubble to the total area of the reaction chamber, the result is:

n.sub.i+1=n.sub.i(1+η(1−V.sub.B,i))

[0067] In the event of a presence of a bubble, the actual number of new DNA strand segments is thus below the assumed number of copied DNA strand segments. For each cycle step, it is then possible to calculate a reaction efficiency:

[00001]$r_i=\frac{n_{i+1}}{n_i}$

[0068] In step S14, the thus determined reaction efficiency in the cycle is used as a scaling factor and the qPCR curve is constructed with the aid of the corrected intensity values n.sub.actual, i from the measured intensity values n.sub.curve, i:

[00002]$n_{\mathrm{actual},i}=\frac{n_{\mathrm{curve},i}}{r_i}$

[0069] In step S15, a check is made as to whether the measurement method should be terminated. For example, this may be the case after a termination criterion has been reached, such as, for example, a specified number of measurement cycles. If this is not the case (alternative: no), the method is continued in step S12, otherwise the method is ended with step S16 and the corrected qPCR curve is evaluated.

[0070] The evaluation in step S16 can be done by classification of the corrected qPCR curve with the aid of a specified classification model. This can determine whether the corrected qPCR curve indicates a presence or a nonpresence of the DNA strand segment to be detected, i.e., indicates whether the DNA strand segment to be detected is present in the substance or not.

[0071] FIG. 8 depicts a flowchart to illustrate a further method which is executed in the control unit. The method can be implemented in a data processing device as hardware and/or software.

[0072] In step S21, the qPCR method is started.

[0073] In step S22, the denaturation step S1 is carried out in the corresponding denaturation reaction chamber.

[0074] In step S23, a brightness h.sub.D of the fluorescent light is recorded at the end of denaturation by the corresponding camera 14 of the denaturation chamber 11.

[0075] In step S24, the annealing process of step S2 is started.

[0076] In step S25, a brightness h.sub.A of the fluorescent light is recorded at the end of the annealing process by the corresponding camera 15 of the annealing chamber 12.

[0077] In step S26, the reaction liquid is conducted into the elongation chamber 13.

[0078] In step S27, a brightness h.sub.E,A of the fluorescent light is recorded at the start of the elongation process by the corresponding camera 16 of the elongation chamber 13.

[0079] In step S28, the elongation process is started.

[0080] In step S29, a brightness h.sub.e,e,i of the fluorescent light is recorded at the end of the elongation step by the corresponding camera 16 of the elongation chamber 13.

[0081] Theoretically, the values h.sub.d,i, h.sub.a,i, h.sub.e,a,i have the same brightness, since no amplification is taking place, and only differ from the brightness value h.sub.e,e,i if additional fluorescence results from the amplification of the elongation process.

[0082] In what follows, a total reaction efficiency is calculated in step S30.

[0083] Owing to bubble formation differing in bubble size, the brightnesses in the various reaction chambers 11, 12, 13 can vary, however. This variation can be used as an indicator for whether bubbles have formed and to what extent. Owing to bubble formation, the reaction liquid is displaced and the efficiency of the individual substeps is thus reduced, since the entire reaction liquid is not available for the reaction. Therefore, bubble volume quotients q as quotients of the brightnesses between the individual substeps can be determined as follows:

[00003]$q_{d,i+1}=\frac{h_{d,i+1}}{h_{e,e,i}}{q}_{a,i}=\frac{h_{a,i}}{h_{d,i}}{q}_{e,i}=\frac{h_{e,a,i}}{h_{a,i}}\eta =\frac{h_{e,e,i}}{h_{e,a,i}}$

[0084] If one of the quotients q should be greater than 1, it is set to 1, since the reaction liquid not processed in the previous step cannot be further processed in the next substep.

[0085] What therefore arises for each substep is the quantity of processed DNA strand segments (n.sub.d,i for the number of DNA strand segments after the denaturation in the i-th cycle, n.sub.a,i for the number of DNA strand segments after the annealing in the i-th cycle, n.sub.e,i for the number of DNA strand segments after the elongation in the i-th cycle)

n.sub.d,i=n.sub.i.Math.q.sub.d,i

n.sub.a,i=n.sub.i.Math.q.sub.a,i

n.sub.e,i=n.sub.i.Math.q.sub.e,i

[0086] The number of DNA strand segments available for elongation and incorporation of fluorescence as at the start of the last substep therefore corresponds to

n.sub.e,a,i=n.sub.i.Math.q.sub.d,i.Math.q.sub.a,i.Math.q.sub.e,i

[0087] Assuming that denatured and elongated DNA strand segments which are not amplified are joined together again to form the original double strands, what results after the PCR cycle, i.e., at the end of the elongation phase, is

n.sub.i+1=n.sub.i(1+η.Math.q.sub.d,i.Math.q.sub.a,i.Math.q.sub.e,i)

as the quantity of DNA strand segments present in total in the reaction liquid.

[0088] In the event of the presence of a bubble in the reaction chamber, the actual number of new DNA strand segments is thus below the theoretically assumed number of DNA strand segments.

[0089] For each cycle step, it is then possible to calculate a total reaction efficiency

[00004]$r_i=\frac{n_{i+1}}{n_i}$

[0090] This reaction efficiency can, as in the exemplary embodiment of FIG. 6, be used as a scaling factor for the measured intensity value at the end of the elongation phase.

[0091] In step S31, the thus determined reaction efficiency in the cycle is used as a scaling factor and the qPCR curve is constructed with the aid of the corrected intensity values n.sub.actual,i from the measured intensity values n.sub.curve,i:

[00005]$n_{\mathrm{actual},i}=\frac{n_{\mathrm{curve},i}}{r_i}$

[0092] In step S32, a check is made as to whether the measurement method should be terminated. For example, this may the case after a termination criterion has been reached, such as, for example, a specified number of measurement cycles. If this is not the case (alternative: no), the method is continued in step S22, otherwise the method is ended with step S33 and the corrected qPCR curve is evaluated.

[0093] The evaluation in step S33 can be done by classification of the corrected qPCR curve with the aid of a specified classification model. This can determine whether the corrected qPCR curve indicates a presence or a nonpresence of the DNA strand segment to be detected, i.e., indicates whether the DNA strand segment to be detected is present in the substance or not.