Nucleic acid detection result evaluation method and device and storage medium

Abstract

The invention provides a nucleic acid detection result evaluation method and device and a storage medium. The nucleic acid detection result evaluation method comprises the following steps: acquiring N original fluorescence values in a nucleic acid cyclic amplification reaction; the N original fluorescence values are preprocessed to obtain N fluorescence values to be processed, and each fluorescence value to be processed in the N fluorescence values to be processed is in a preset range; according to the to-be-processed fluorescence value of the ith cycle and the to-be-processed fluorescence value of the (i-1) th cycle, the fluorescence change value of the ith cycle is determined to obtain N fluorescence change values, i is larger than 1 and smaller than or equal to N, and the fluorescence change value of the first cycle is a preset value; and determining whether the nucleic acid detection result of the nucleic acid cyclic amplification reaction is positive or not according to the maximum value in the N fluorescence change values. According to the invention, the sensitivity, robustness and universality of nucleic acid detection can be improved.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of nucleic acid detection, and in particular to a nucleic acid detection result evaluation method and device and a storage medium. BACKGROUND

[0002] NAT (Nucleic Acid Testing) is one of the important methods of molecular diagnosis, and is widely used for detection of low-concentration target sequences due to its multiple detection capability and high sensitivity. qPCR (Quantitative Polymerase Chain Reaction) technology is a nucleic acid detection technology developed on the basis of PCR (Polymerase Chain Reaction) technology, and is one of the commonly used nucleic acid detection technologies.

[0003] qPCR technology realizes exponential amplification of target sequences through specific primers, and quantifies target sequences by detecting the intensity of fluorescence signals with the help of fluorescence probes combined with target sequences, so as to obtain nucleic acid detection results. SUMMARY

[0004] The inventors have noticed that in the related art, nucleic acid detection is performed by using qPCR technology, and the detection results are affected by many complex factors, such as pathogen nucleic acid sequence variation, sample initial concentration, and reaction condition difference, so that the robustness and universality of nucleic acid detection cannot be guaranteed.

[0005] Accordingly, the present disclosure provides a nucleic acid detection result evaluation method, which can effectively improve the sensitivity, robustness and universality of nucleic acid detection.

[0006] According to a first aspect of an embodiment of the present disclosure, a nucleic acid detection result evaluation method is provided, comprising: obtaining N original fluorescence values in a nucleic acid cycle amplification reaction; preprocessing the N original fluorescence values to obtain N to-be-processed fluorescence values, wherein each to-be-processed fluorescence value in the N to-be-processed fluorescence values is within a predetermined range; determining a fluorescence change value of the ith cycle according to the to-be-processed fluorescence value of the ith cycle and the to-be-processed fluorescence value of the (i-1)th cycle, to obtain N fluorescence change values, 1

[0007] In some embodiments, determining whether the nucleic acid detection result of the nucleic acid cycle amplification reaction is positive according to the maximum value in the N fluorescence change values comprises: if the maximum value in the N fluorescence change values is greater than a predetermined threshold value, determining that the nucleic acid detection result of the nucleic acid cycle amplification reaction is positive.

[0008] In some embodiments, if the maximum value of the N fluorescence change values is greater than the predetermined threshold value, determining the nucleic acid detection result of the nucleic acid cycle amplification reaction as positive comprises: if the maximum value of the N fluorescence change values is greater than the predetermined threshold value, querying the cycle number M corresponding to the maximum value; if the fluorescence change value of the first cycle to the fluorescence change value of the Mth cycle shows an upward trend, and the fluorescence change value of the Mth cycle to the fluorescence change value of the Nth cycle shows a downward trend, determining the nucleic acid detection result as positive.

[0009] In some embodiments, determining the fluorescence change value of the ith cycle according to the to-be-processed fluorescence value of the ith cycle and the to-be-processed fluorescence value of the i-1th cycle comprises: determining the fluorescence change value of the ith cycle according to the ratio of the to-be-processed fluorescence value of the ith cycle and the to-be-processed fluorescence value of the i-1th cycle.

[0010] In some embodiments, preprocessing the N original fluorescence values comprises: filtering the N original fluorescence values to obtain N intermediate values; and normalizing the N intermediate values to obtain the N to-be-processed fluorescence values.

[0011] In some embodiments, further comprising: determining a fluorescence reference value in the N to-be-processed fluorescence values; determining a linear fluorescence growth function according to the fluorescence reference value; determining a first fluorescence accumulation amount in an interval from a candidate cycle number to a cycle number corresponding to the fluorescence reference value, and determining a second fluorescence accumulation amount in an interval from the cycle number corresponding to the fluorescence reference value to a cycle number corresponding to a maximum to-be-processed fluorescence value in the N to-be-processed fluorescence values, according to the linear fluorescence growth function; determining a first energy transfer value of a distance between a donor and an acceptor in an interval from a specified distance to a first distance, and determining a second energy transfer value of the distance between the donor and the acceptor in an interval from a second distance to the specified distance, according to a transfer efficiency function of fluorescence resonance energy transfer (FRET), wherein the specified distance is a distance between the donor and the acceptor when the transfer efficiency of FRET is 50%; calculating a ratio of the first fluorescence accumulation amount and the second fluorescence accumulation amount as a first ratio; calculating a ratio of the first energy transfer value and the second energy transfer value as a second ratio; and in the case that the first ratio is equal to the second ratio, taking the candidate cycle number as a threshold cycle number.

[0012] In some embodiments, determining the fluorescence reference value in the N to-be-processed fluorescence values comprises: identifying a baseline region and a linear region in the N to-be-processed fluorescence values; obtaining a fluorescence background value from the baseline region; and calculating a median value of a maximum to-be-processed fluorescence value in the N to-be-processed fluorescence values and the fluorescence background value as the fluorescence reference value, wherein the fluorescence reference value is located in the linear region.

[0013] In some embodiments, obtaining the fluorescence background value from the baseline region comprises: taking any to-be-processed fluorescence value in the baseline region as the fluorescence background value; or taking an average of all to-be-processed fluorescence values in the baseline region as the fluorescence background value; or taking a to-be-processed fluorescence value corresponding to a maximum cycle number in the baseline region as the fluorescence background value.

[0014] In some embodiments, determining the linear fluorescence growth function according to the fluorescence reference value comprises: fitting the fluorescence reference value and a plurality of to-be-processed fluorescence values adjacent to the fluorescence reference value in the linear region to obtain the linear fluorescence growth function.

[0015] In some embodiments, determining the first fluorescence accumulation amount in the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value comprises: calculating an integral value in the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value according to the linear fluorescence growth function as the first fluorescence accumulation amount; and determining the second fluorescence accumulation amount in the interval from the cycle number corresponding to the fluorescence reference value to the cycle number corresponding to the maximum to-be-processed fluorescence value in the N to-be-processed fluorescence values comprises: calculating an integral value in the interval from the cycle number corresponding to the fluorescence reference value to the cycle number corresponding to the maximum to-be-processed fluorescence value according to the linear fluorescence growth function as the second fluorescence accumulation amount.

[0016] In some embodiments, determining the first energy transfer value when the distance between the donor and the acceptor is in the interval from the specified distance to the first distance comprises: calculating an integral value when the distance between the donor and the acceptor is in the interval from the specified distance to the first distance according to the transfer efficiency function of FRET as the first energy transfer value; and determining the second energy transfer value when the distance between the donor and the acceptor is in the interval from the second distance to the specified distance comprises: calculating an integral value when the distance between the donor and the acceptor is in the interval from the second distance to the specified distance according to the transfer efficiency function of FRET as the second energy transfer value.

[0017] In some embodiments, the difference between the first distance and the specified distance is a specified distance difference value; and the difference between the specified distance and the second distance is the specified distance difference value.

[0018] In some embodiments, the specified distance difference value is a specified multiple of the specified distance.

[0019] In some embodiments, the specified multiple is 0.5.

[0020] According to a second aspect of the embodiments of the present disclosure, a nucleic acid detection result evaluation device is provided, comprising: a memory; a processor coupled to the memory, the processor being configured to execute instructions stored in the memory to implement the nucleic acid detection result evaluation method according to any of the above embodiments.

[0021] According to a third aspect of the embodiments of the present disclosure, a computer readable storage medium is provided, wherein the computer readable storage medium stores computer instructions, and the computer instructions are executed by a processor to implement the nucleic acid detection result evaluation method according to any one of the above embodiments.

[0022] According to a fourth aspect of the embodiments of the present disclosure, a computer program product is provided, comprising computer instructions, and the computer instructions are executed by a processor to implement the nucleic acid detection result evaluation method according to any one of the above embodiments.

[0023] Other features and advantages of the present disclosure will be apparent from the following detailed description of exemplary embodiments of the present disclosure, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can be obtained by those skilled in the art without creative labor under the premise of the drawings.

[0025] Figure 1 A flowchart of the nucleic acid detection result evaluation method according to an embodiment of the present disclosure;

[0026] Figures 2 to 5 A schematic diagram of the amplification curve and the corresponding fluorescence change value of the negative sample according to some embodiments of the present disclosure;

[0027] Figure 6 A schematic diagram of the amplification curve and the corresponding fluorescence change value of the positive sample according to an embodiment of the present disclosure;

[0028] Figure 7 A flowchart of the nucleic acid detection result evaluation method according to another embodiment of the present disclosure;

[0029] Figure 8 A schematic diagram of the amplification curve and the corresponding FRET transfer efficiency curve of the positive sample according to an embodiment of the present disclosure;

[0030] Figure 9 A structural schematic diagram of the nucleic acid detection result evaluation device according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present disclosure will be described clearly and completely in the embodiments of the present disclosure in combination with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. The description of the at least one example embodiment is actually only illustrative, but not as any limitation on the present disclosure and its application or use. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present disclosure.

[0032] Unless specifically stated, the relative arrangement of the components and steps, numerical expressions, and values shown in these embodiments do not limit the scope of the present disclosure.

[0033] At the same time, it should be understood that, for the convenience of description, the sizes of the various parts shown in the drawings are not drawn in accordance with the actual proportional relationship.

[0034] The techniques, methods, and devices known to those of ordinary skill in the relevant art can not be discussed in detail, but should be considered as part of the authorized description where appropriate.

[0035] In all examples shown and discussed herein, any specific values should be interpreted as merely illustrative, and not as a limitation. Thus, other examples of example embodiments can have different values.

[0036] It should be noted that: similar reference numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0037] The inventors have noticed that the qPCR detection currently faces the following problems.

[0038] First, the detection results are easily affected by many complex factors, such as pathogen DNA sequence variation, sample initial concentration, reaction condition difference, and qPCR inhibitors, making it difficult to evaluate the relationship between fluorescence value and target sequence replication rate.

[0039] Second, in large-scale pathogen detection, rapid mutation of viruses leads to more obvious geographical differences in viruses, further increasing the difficulty of detection, and failing to effectively maintain the sensitivity, robustness and universality of the detection results.

[0040] Accordingly, the present disclosure provides a nucleic acid detection result evaluation method, which can effectively improve the sensitivity, robustness and universality of nucleic acid detection, which has great significance for the application of nucleic acid detection in major disease diagnosis, prognosis treatment and infectious disease epidemic control.

[0041] Figure 1 A flowchart of a nucleic acid detection result evaluation method for an embodiment of the present disclosure is shown. In some embodiments, the following nucleic acid detection result evaluation method is performed by a nucleic acid detection result evaluation device, including steps 11-14.

[0042] In step 11, N original fluorescence values in a nucleic acid cycle amplification reaction are obtained, where N is a natural number greater than 1.

[0043] It should be noted that during the nucleic acid cycle amplification reaction, an original fluorescence value is collected at each cycle to draw an amplification curve, which reflects the change of fluorescence signal with the number of cycles. The amplification curve can generally be divided into four regions: baseline region, exponential region, linear region and plateau region.

[0044] In step 12, the N original fluorescence values are preprocessed to obtain N to-be-processed fluorescence values, wherein each to-be-processed fluorescence value in the N to-be-processed fluorescence values is within a predetermined range.

[0045] It should be noted that preprocessing the original fluorescence values can effectively improve the accuracy of the nucleic acid detection result. Preprocessing can generally include reducing background noise, normalizing data, and removing outliers, etc.

[0046] In some embodiments, preprocessing the N original fluorescence values includes filtering the N original fluorescence values to obtain N intermediate values. Next, the N intermediate values are normalized to obtain the N to-be-processed fluorescence values.

[0047] It should be noted that noise usually exists in the form of sharp peak mutation or small amplitude fluctuation, and noise will affect the accuracy of nucleic acid detection. Curve smoothing helps to reduce sharp peak noise and fluctuation noise, and improve the signal-to-noise ratio of fluorescence data. Different filtering methods are needed due to different curve characteristics. For example, the fluorescence value of sharp peak noise differs greatly from the adjacent value, which is difficult to correct by weighted or optimal estimation, and median filtering has good denoising effect on it, while preserving the data edge information and curve detail characteristics. For example, fluctuation noise fluctuates slightly in a small range, which can be smoothed and weakened by mean filtering combined with other fluorescence values in the neighborhood.

[0048] In addition, at the end of the nucleic acid cycle amplification reaction, when the raw materials required for amplification, such as primers, enzymes, etc., have been consumed, lost activity or the production and quenching of fluorescence have reached a balanced state, the amplification curve enters the platform region. In large-scale nucleic acid detection, due to random interference in the amplification reaction, even if the sample size and reagent amount are the same, the fluorescence signal intensity reached by the platform region of the amplification curve of different samples may be different. In order to more intuitively compare and analyze the data of different samples, the data can be normalized. For example, the fluorescence value is adjusted to a predetermined range by scaling by a maximum amplitude value of 1.

[0049] In step 13, the fluorescence change value of the i th cycle is determined according to the to-be-processed fluorescence value of the i th cycle and the to-be-processed fluorescence value of the i-1 th cycle, to obtain N fluorescence change values, 1 < i ≤ N, wherein the fluorescence change value of the 1 st cycle is a predetermined value.

[0050] In some embodiments, the fluorescence change value of the i th cycle is determined according to the ratio of the to-be-processed fluorescence value of the i th cycle and the to-be-processed fluorescence value of the i-1 th cycle.

[0051] For example, assuming that the to-be-processed fluorescence value of the i th cycle is F(i), and the to-be-processed fluorescence value of the i-1 th cycle is F(i-1), then the fluorescence change value DD(i) of the i th cycle is shown in formula (1).

[0052] DD(i) = F(i) / F(i-1) (1)

[0053] For example, the fluorescence change value of the 1 st cycle can be determined as 1. That is, the value of DD(1) is 1.

[0054] In step 14, whether the nucleic acid detection result of the nucleic acid cycle amplification reaction is positive is determined according to the maximum value of the N fluorescence change values.

[0055] It should be noted that since the above embodiment determines whether the nucleic acid detection result is positive based on the maximum value of the N fluorescence change values, it can also be referred to as a qualitative analysis method.

[0056] In some embodiments, if the maximum value of the N fluorescence change values is greater than a predetermined threshold value, it is determined that the nucleic acid detection result of the nucleic acid cycle amplification reaction is positive.

[0057] It should be noted that for a negative sample of nucleic acid detection, the fluorescence change value presents irregular fluctuations around 1. For a positive sample of nucleic acid detection, the fluorescence change value will have a peak value in the region where the to-be-processed fluorescence value increases significantly. Therefore, if the maximum value of the N fluorescence change values is greater than a predetermined threshold value, it can be considered that the corresponding nucleic acid detection result is positive.

[0058] In some embodiments, if the maximum value among the N fluorescence change values ​​is greater than a predetermined threshold, the cycle number M corresponding to the maximum value is further queried. If the fluorescence change value from the first cycle to the Mth cycle shows an upward trend, and the fluorescence change value from the Mth cycle to the Nth cycle shows a downward trend, then the nucleic acid test result is determined to be positive.

[0059] It's important to note that for positive nucleic acid test samples, the initial copy number of the target sequence is higher, resulting in a faster rate of nucleic acid cyclic amplification and a greater rate of fluorescence signal growth during the reaction. Therefore, for positive nucleic acid test samples, the fluorescence signal intensity increases exponentially in the exponential region of the amplification curve, then maintains a stable increase in the linear region, and tends to plateau in the plateau region. In other words, in positive samples, the change in fluorescence signal intensity exhibits a characteristic of first increasing and then decreasing.

[0060] Therefore, if multiple fluorescence change values ​​show an overall trend of first increasing and then decreasing, and the maximum value among these multiple fluorescence change values ​​is greater than a predetermined threshold, then the nucleic acid test result is determined to be positive.

[0061] For negative samples in nucleic acid testing, the initial copy number of the target sequence is extremely low, even zero, resulting in a slow rate of nucleic acid cyclic amplification and a smaller rate of fluorescence signal growth during the reaction. Therefore, negative samples typically do not exhibit obvious exponential, linear, or plateau regions, and the change in fluorescence signal intensity does not show a characteristic of first increasing and then decreasing.

[0062] The above embodiments are illustrated below with specific examples.

[0063] exist Figure 2 middle, Figure 2 a is the amplification curve of a negative sample, where Figure 2 The x-axis of 'a' represents the cycle number, and the y-axis represents the fluorescence value. Figure 2 b is the result of using the above formula (1) to... Figure 2 The amplification curve shown in figure a was processed to obtain multiple fluorescence change values, among which... Figure 2 The x-axis of b represents the cycle number, and the y-axis represents the fluorescence change value.

[0064] from Figure 2 As can be seen, the baseline, exponential, linear, and plateau regions cannot be identified from the amplification curve. Figure 2 As can be seen from b, the fluorescence change value fluctuates irregularly around 1, and the maximum value of the fluorescence change value is also significantly less than 1.01.

[0065] exist Figure 3 middle,Figure 3 a is the amplification curve of another negative sample, where Figure 3 The x-axis of 'a' represents the cycle number, and the y-axis represents the fluorescence value. Figure 3 b is the result of using the above formula (1) to... Figure 3 The amplification curve shown in figure a was processed to obtain multiple fluorescence change values, among which... Figure 3 The x-axis of b represents the cycle number, and the y-axis represents the fluorescence change value.

[0066] from Figure 3 As can be seen, the baseline, exponential, linear, and plateau regions cannot be identified from the amplification curve. Figure 3 As can be seen from b, the fluorescence change value fluctuates irregularly around 1, and the maximum value of the fluorescence change value does not exceed 1.025.

[0067] exist Figure 4 middle, Figure 4 a is the amplification curve of another negative sample, where Figure 4 The x-axis of 'a' represents the cycle number, and the y-axis represents the fluorescence value. Figure 4 b is the result of using the above formula (1) to... Figure 4 The amplification curve shown in figure a was processed to obtain multiple fluorescence change values, among which... Figure 4 The x-axis of b represents the cycle number, and the y-axis represents the fluorescence change value.

[0068] from Figure 4 As can be seen, the baseline, exponential, linear, and plateau regions cannot be identified from the amplification curve. Figure 4 As can be seen from b, the fluorescence change value fluctuates irregularly around 1, and the maximum value of the fluorescence change value does not exceed 1.01.

[0069] exist Figure 5 middle, Figure 5 a is the amplification curve of another negative sample, where Figure 5 The x-axis of 'a' represents the cycle number, and the y-axis represents the fluorescence value. Figure 5 b is the result of using the above formula (1) to... Figure 5 The amplification curve shown in figure a was processed to obtain multiple fluorescence change values, among which... Figure 5 The x-axis of b represents the cycle number, and the y-axis represents the fluorescence change value.

[0070] from Figure 5 As can be seen, the baseline, exponential, linear, and plateau regions cannot be identified from the amplification curve. Figure 5 As can be seen from b, the fluorescence change value fluctuates irregularly around 1, and the maximum value of the fluorescence change value does not exceed 1.01.

[0071] exist Figure 6 middle,Figure 6 a is an amplification curve of a positive sample, wherein Figure 6 a is a graph with cycle number as the horizontal coordinate and fluorescence value as the vertical coordinate. Figure 6 b is a plurality of fluorescence change values obtained by processing the amplification curve shown in a using the above formula (1), wherein Figure 6 b is a graph with cycle number as the horizontal coordinate and fluorescence change value as the vertical coordinate. Figure 6

[0072] As can be seen from a, the baseline region, the exponential region, the linear region and the plateau region can be clearly identified from the amplification curve. As can be seen from b, the curve formed by the plurality of fluorescence change values has a sharp peak, and the maximum value in the plurality of fluorescence change values is close to 1.10. In addition, the plurality of fluorescence change values as a whole presents a trend of first rising and then falling. Figure 6 Figure 6 As can be seen from the example of a, by using the qualitative analysis method provided by the above embodiment, the sensitivity, robustness and universality of nucleic acid detection can be effectively improved.

[0073] As can be seen from the example of a, by using the qualitative analysis method provided by the above embodiment, the sensitivity, robustness and universality of nucleic acid detection can be effectively improved. Figures 2 to 6

[0074] Figure 7 FIG. 1 is a flowchart of a nucleic acid detection result evaluation method according to an embodiment of the present disclosure. In some embodiments, the nucleic acid detection result evaluation method comprises steps 1-7.

[0075] In step 1, a fluorescence reference value in N fluorescence values to be processed is determined.

[0076] In some embodiments, the step of determining the fluorescence reference value in the N fluorescence values to be processed comprises the following contents.

[0077] 1) Identify the baseline region and the linear region in the N fluorescence values to be processed.

[0078] 2) Obtain a fluorescence background value from the baseline region.

[0079] In some embodiments, any fluorescence value to be processed in the baseline region is taken as the fluorescence background value. Alternatively, the average value of all fluorescence values to be processed in the baseline region is taken as the fluorescence background value. Alternatively, the fluorescence value to be processed corresponding to the maximum cycle number in the baseline region is taken as the fluorescence background value.

[0080] It should be noted that since the baseline region is mainly composed of background noise, no matter which way is used to obtain the fluorescence background value, it will not affect the final evaluation result.

[0081] ​​​3) calculating the median of the maximum to-be-processed fluorescence value in the N to-be-processed fluorescence values and the fluorescence background value as a fluorescence reference value, wherein the fluorescence reference value is in the linear region.

[0082] At step 72, a linear fluorescence growth function is determined according to the fluorescence reference value.

[0083] In some embodiments, the linear fluorescence growth function is fitted using the fluorescence reference value and a plurality of to-be-processed fluorescence values adjacent to the fluorescence reference value. The linear fluorescence growth function is used to represent the case of the intensity of the fluorescence signal growing with time.

[0084] It should be noted that in the linear region of the amplification curve, the growth of the fluorescence signal intensity is relatively stable and linearly related to the cycle number. Therefore, in order to obtain an accurate linear fluorescence growth function, the plurality of to-be-processed fluorescence values adjacent to the fluorescence reference value used in the fitting process are also in the linear region of the amplification curve. In addition, the fluorescence reference value is on the straight line formed by the linear fluorescence growth function.

[0085] For example, the fitting method can use linear regression methods such as least squares method, Bayesian linear regression, ridge regression, etc.

[0086] At step 73, a first fluorescence accumulation amount in the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value is determined according to the linear fluorescence growth function, and a second fluorescence accumulation amount in the interval from the cycle number corresponding to the fluorescence reference value to the cycle number corresponding to the maximum to-be-processed fluorescence value in the N to-be-processed fluorescence values is determined.

[0087] In some embodiments, an integral value in the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value is calculated as the first fluorescence accumulation amount according to the linear fluorescence growth function. An integral value in the interval from the cycle number corresponding to the fluorescence reference value to the cycle number corresponding to the maximum to-be-processed fluorescence value is calculated as the second fluorescence accumulation amount according to the linear fluorescence growth function.

[0088] For example, assuming that the linear fluorescence growth function is f(x), the candidate cycle number is x0, the cycle number corresponding to the fluorescence reference value is x Mid , and the cycle number corresponding to the maximum to-be-processed fluorescence value is x Max , then the first fluorescence accumulation amount S1 is shown in formula (2), and the second fluorescence accumulation amount S2 is shown in formula (3).

[0089]

[0090]

[0091] At step 74, a first energy transfer value is determined according to a transfer efficiency function of FRET (Fluorescence Resonance Energy Transfer) for a distance between the donor and the acceptor being in an interval from a specified distance to a first distance, and a second energy transfer value is determined according to the transfer efficiency function of FRET for a distance between the donor and the acceptor being in an interval from a second distance to the specified distance, where the specified distance is a distance between the donor and the acceptor at which the transfer efficiency of FRET is 50%.

[0092] It is noted herein that FRET is a photophysical process that depends on the distance between the donor and acceptor molecules, in which an excited-state fluorophore (the donor) transfers energy nonradiatively to a non-adjacent acceptor molecule through dipole-dipole interactions, resulting in quenching of donor fluorescence and an increase in acceptor fluorescence emission.

[0093] The transfer efficiency E(r) of FRET with respect to the distance r between the donor and the acceptor is shown in equation (4).

[0094]

[0095] where R0 represents the distance between the donor and the acceptor at which the transfer efficiency of FRET is 50%. That is, the specified distance is R0.

[0096] In some embodiments, an integral value is calculated according to the transfer efficiency function of FRET for a distance between the donor and the acceptor being in an interval from the specified distance to the first distance as the first energy transfer value. In addition, an integral value is calculated according to the transfer efficiency function of FRET for a distance between the donor and the acceptor being in an interval from the second distance to the specified distance as the second energy transfer value.

[0097] In some embodiments, the difference between the first distance and the specified distance is a specified distance difference value, and the difference between the specified distance and the second distance is the specified distance difference value. That is, the difference between the first distance and the specified distance is equal to the difference between the specified distance and the second distance.

[0098] In some embodiments, the specified distance difference value is a specified multiple of the specified distance.

[0099] For example, the specified distance difference value is 0.5 times the specified distance. That is, the specified distance is R0, the first distance is 1.5R0, and the second distance is 0.5R0, so that the difference between the first distance and the specified distance is 0.5R0, and the difference between the specified distance and the second distance is 0.5R0. In this case, the first energy transfer value E1 is shown in equation (5), and the second energy transfer value E2 is shown in equation (6).

[0100]

[0101]

[0102] At step 75, the ratio of the first fluorescence accumulation amount and the second fluorescence accumulation amount is calculated as a first ratio.

[0103] At step 76, the ratio of the first energy transfer value and the second energy transfer value is calculated as a second ratio.

[0104] At step 77, in the case where the first ratio is equal to the second ratio, the candidate cycle number is taken as a threshold cycle number (Ct value).

[0105] That is, if formula (7) is satisfied, the candidate cycle number x0 is taken as the threshold cycle number.

[0106]

[0107] By substituting formula (2), formula (3), formula (5) and formula (6) into formula (7), the following formula (8) can be obtained.

[0108]

[0109] That is, the candidate cycle number x0 satisfying formula (8) is taken as the threshold cycle number.

[0110] It should be noted that the threshold cycle number refers to the cycle number at which the fluorescence signal first significantly exceeds the background signal during the qPCR reaction. According to the threshold cycle number, the nucleic acid detection result can be accurately evaluated.

[0111] The above embodiments will be described below through specific examples.

[0112] Figure 8 A schematic diagram of the amplification curve and the corresponding FRET transfer efficiency curve of a positive sample of an embodiment of the present disclosure.

[0113] In the Figure 8 , curve 81 is the FRET transfer efficiency curve of a positive sample, and in the coordinate 801 corresponding to curve 81, the horizontal coordinate is the distance, and the vertical coordinate is the transfer efficiency. Curve 82 is the amplification curve of a positive sample, and in the coordinate 802 corresponding to curve 82, the horizontal coordinate is the cycle number, and the vertical coordinate is the fluorescence value. Curve 83 is a linear fluorescence growth function curve determined according to the fluorescence reference value, and curve 83 corresponds to the same coordinate 802 as curve 82.

[0114] As Figure 8 shown, R0 represents the distance between the donor and the acceptor when the transfer efficiency of FRET is 50%. As the PCR amplification reaction proceeds, the number of fluorescence probes decreases with the increase of the target gene, and therefore the transfer efficiency of FRET decreases accordingly.

[0115] Using the above formula (5), the integral value of the distance between the donor and the recipient in the interval from R0 to 1.5R0 is calculated based on curve 81, and is used as the first energy transfer value E1. In addition, using the above formula (6), the integral value of the distance between the donor and the recipient in the interval from 0.5R0 to R0 is calculated based on curve 81, and is used as the second energy transfer value E2.

[0116] like Figure 8 As shown, the cycle number corresponding to the maximum untreated fluorescence value in curve 82 is x. Max The median of the maximum fluorescence value to be treated and the fluorescence background value in curve 82 is calculated as the fluorescence reference value, where the cycle number corresponding to the fluorescence reference value is x. Mid Using a fluorescence reference value and multiple adjacent fluorescence values, a linear fluorescence growth function curve 83 is obtained. Let the candidate cycle number be x0. Using the above formula (2), the value of the fluorescence growth function between x0 and x3 is calculated based on curve 83. Mid The integral value within the interval is taken as the first fluorescence accumulation S1. Using the above formula (3), the value at x is calculated according to curve 83. Mid To x Max The integral value within the interval is used as the second fluorescence accumulation S2.

[0117] Next, using the above formula (7) or formula (8), the x0 that meets the requirements is taken as the threshold loop number.

[0118] It should be noted here that, compared to the qualitative analysis method described above, Figure 7 The embodiment obtains the threshold cycle number through quantitative calculation, therefore it can also be used to... Figure 7 The aforementioned embodiment is referred to as the quantitative analysis method.

[0119] In the nucleic acid detection result evaluation method provided in the above embodiments of this disclosure, the fluorescence accumulation in a specified region is calculated using the amplification curve, and the energy transfer value in the specified region is calculated using the FRET transfer efficiency curve. This allows for accurate calculation of the threshold cycle number, enabling precise quantitative analysis of the sample. This method is applicable to large-scale nucleic acid detection and improves the robustness and universality of the nucleic acid detection result evaluation method.

[0120] Figure 9 This is a schematic diagram of the structure of a nucleic acid detection result evaluation device according to an embodiment of this disclosure. Figure 9 As shown, the nucleic acid test result evaluation device 90 includes a memory 91, a processor 92, and a bus 93 connecting different system components.

[0121] The memory 91 can include, for example, a system memory, a non-volatile storage medium, and the like. The system memory stores, for example, an operating system, application programs, a Boot Loader, and other programs. The system memory can include a volatile storage medium such as a random access memory (RAM) and / or a cache memory. The non-volatile storage medium stores, for example, instructions of at least one nucleic acid test result evaluation method being executed. The non-volatile storage medium includes, but is not limited to, a magnetic disk storage, an optical disk storage, a flash memory, and the like.

[0122] The processor 92 can be implemented in a manner of a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, a discrete gate or transistor, and the like discrete hardware component. Accordingly, the method in any of the above embodiments can be implemented by a central processing unit (CPU) running instructions of corresponding steps stored in the memory, or by a dedicated circuit performing corresponding steps.

[0123] For example, the processor 92 is configured to perform the method as described in any of Figure 1 or Figure 7 embodiments based on instructions stored in the memory.

[0124] The bus 93 can use any of a variety of bus structures. For example, the bus structure includes, but is not limited to, an industry standard architecture (ISA) bus, a microchannel architecture (MCA) bus, a peripheral component interconnect (PCI) bus.

[0125] The interfaces 94, 95, 96 of the nucleic acid test result evaluation apparatus 90, and the memory 91 and the processor 92 can be connected through the bus 93. The input / output interface 94 can provide a connection interface for input / output devices such as a display, a mouse, a keyboard, and the like. The network interface 95 provides a connection interface for various networking devices. The storage interface 96 provides a connection interface for external storage devices such as a floppy disk, a U disk, an SD card, and the like.

[0126] Here, various aspects of the present disclosure are described with reference to flowcharts and / or block diagrams of the methods, apparatuses, and computer program products according to embodiments of the present disclosure. It should be understood that each block of the flowcharts and / or block diagrams, and combinations of blocks, can be implemented by computer readable program instructions.

[0127] These computer readable program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable device to produce a machine, so that the instructions executed by the processor produce the apparatus that implements the functions specified in one or more blocks of the flowcharts and / or block diagrams.

[0128] These computer readable program instructions can also be stored in a computer readable storage medium that can be static or dynamic. The computer readable storage medium is a tangible storage medium that can be used for storing data and / or instructions that can be read by a computer system. The computer readable storage medium can be volatile (such as random access memory (RAM), etc.) or non-volatile (such as read-only memory (ROM), floppy disks, etc.). The computer readable storage medium can also be other types of tangible storage medium, for example, a compact disc read-only memory (CD-ROM), tape, etc.

[0129] The present disclosure can take the form of an entirely hardware embodiment, an entirely software embodiment or an embodiment containing both software and hardware aspects.

[0130] The present disclosure also provides a computer readable storage medium, wherein the computer readable storage medium stores computer instructions, and the computer instructions are executed by a processor to implement the method as described in any one of Figure 1 or Figure 7 any one of the embodiments.

[0131] The present disclosure also provides a computer program product, comprising computer instructions, and the computer instructions are executed by a processor to implement the method as described in any one of Figure 1 or Figure 7 any one of the embodiments.

[0132] In some embodiments, the functional units described above can be implemented as a general processor, a programmable logic controller (PLC), a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, a discrete gate or transistor logic device, a discrete hardware component, or any appropriate combination thereof for performing the functions described in the present disclosure.

[0133] Those of ordinary skill in the art can understand that all or part of the steps of the above-mentioned embodiments can be completed by hardware, or by program instructing relevant hardware, and the program can be stored in a computer readable storage medium. The storage medium mentioned above can be a read-only memory, a magnetic disk or an optical disk, etc.

[0134] The description of the present disclosure is given for the purpose of illustration and description, and is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. Embodiments are chosen and described in order to best explain the principles of the present disclosure and its practical application, and to enable others skilled in the art to understand the present disclosure for various embodiments with various modifications as are suited to the particular use contemplated.

Claims

1. A method for evaluating nucleic acid detection results, comprising: Obtaining N original fluorescence values in a nucleic acid cyclic amplification reaction; Preprocessing the N original fluorescence values to obtain N fluorescence values to be processed, wherein each fluorescence value to be processed among the N fluorescence values to be processed is within a predetermined range; Determining a fluorescence change value of the i-th cycle according to the fluorescence value to be processed of the i-th cycle and the fluorescence value to be processed of the (i - 1)-th cycle to obtain N fluorescence change values, where 1 < i ≤ N, and the fluorescence change value of the first cycle is a predetermined value; Determining whether the nucleic acid detection result of the nucleic acid cyclic amplification reaction is positive according to the maximum value among the N fluorescence change values.

2. The method for evaluating nucleic acid test results according to claim 1, wherein, The determining whether the nucleic acid detection result of the nucleic acid cyclic amplification reaction is positive according to the maximum value among the N fluorescence change values includes: If the maximum value among the N fluorescence change values is greater than a predetermined threshold, determining that the nucleic acid detection result of the nucleic acid cyclic amplification reaction is positive.

3. The method for evaluating nucleic acid test results according to claim 2, wherein, The if the maximum value among the N fluorescence change values is greater than a predetermined threshold, determining that the nucleic acid detection result of the nucleic acid cyclic amplification reaction is positive includes: If the maximum value among the N fluorescence change values is greater than a predetermined threshold, querying the cycle number M corresponding to the maximum value; If the fluorescence change values from the first cycle to the M-th cycle show an upward trend, and the fluorescence change values from the M-th cycle to the N-th cycle show a downward trend, determining that the nucleic acid detection result is positive.

4. The method for evaluating nucleic acid test results according to claim 1, wherein, The determining the fluorescence change value of the i-th cycle according to the fluorescence value to be processed of the i-th cycle and the fluorescence value to be processed of the (i - 1)-th cycle includes: Determining the fluorescence change value of the i-th cycle according to the ratio of the fluorescence value to be processed of the i-th cycle and the fluorescence value to be processed of the (i - 1)-th cycle.

5. The method for evaluating nucleic acid test results according to claim 1, wherein, The preprocessing the N original fluorescence values includes: Performing a filtering process on the N original fluorescence values to obtain N intermediate values; Performing a normalization process on the N intermediate values to obtain the N fluorescence values to be processed.

6. The method for evaluating nucleic acid detection results according to claim 1, further comprising: Determining a fluorescence reference value among the N fluorescence values to be processed; Determining a linear fluorescence growth function according to the fluorescence reference value; Determining a first fluorescence accumulation amount in the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value according to the linear fluorescence growth function, and determining a second fluorescence accumulation amount in the interval from the cycle number corresponding to the fluorescence reference value to the cycle number corresponding to the maximum fluorescence value to be processed among the N fluorescence values to be processed; Determining a first energy transfer value in the interval from a specified distance to a first distance for the distance between the donor and the acceptor according to the transfer efficiency function of fluorescence resonance energy transfer (FRET), and determining a second energy transfer value in the interval from a second distance to the specified distance for the distance between the donor and the acceptor, wherein the specified distance is the distance between the donor and the acceptor when the transfer efficiency of the FRET is 50%; Calculate the ratio of the first cumulative fluorescence amount to the second cumulative fluorescence amount, and use it as the first ratio value; Calculate the ratio of the first energy transfer value to the second energy transfer value, and use it as the second ratio. If the first ratio is equal to the second ratio, the candidate cycle number is used as the threshold cycle number.

7. The method for evaluating nucleic acid test results according to claim 6, wherein, Determining the fluorescence reference value among the N fluorescence values ​​to be processed includes: Identify the baseline and linear regions among the N fluorescence values ​​to be processed; Obtain fluorescence background values ​​from the baseline region; The median of the maximum fluorescence value to be processed and the fluorescence background value among the N fluorescence values ​​to be processed is calculated as the fluorescence reference value, wherein the fluorescence reference value is located in the linear region.

8. The method for evaluating nucleic acid test results according to claim 7, wherein, The step of obtaining fluorescence background values ​​from the baseline region includes: Take any fluorescence value to be processed in the baseline region as the fluorescence background value; or The average value of all fluorescence values ​​to be processed in the baseline region is taken as the fluorescence background value; or The fluorescence value corresponding to the maximum cycle number in the baseline region is used as the fluorescence background value.

9. The method for evaluating nucleic acid test results according to claim 6, wherein, Determining the linear fluorescence growth function based on the fluorescence reference value includes: A linear fluorescence growth function is obtained by fitting the fluorescence reference value with multiple fluorescence values ​​adjacent to the fluorescence reference value.

10. The method for evaluating nucleic acid detection results according to claim 6, wherein, The determination of the first cumulative fluorescence amount within the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value includes: Based on the linear fluorescence growth function, the integral value within the interval from the candidate cycle number to the cycle number corresponding to the fluorescence reference value is calculated and used as the first fluorescence accumulation. The second fluorescence accumulation amount, determined within the interval from the cycle number corresponding to the fluorescence reference value to the cycle number corresponding to the largest fluorescence value among the N fluorescence values ​​to be processed, includes: Based on the linear fluorescence growth function, the integral value within the interval from the cycle number corresponding to the fluorescence baseline value to the cycle number corresponding to the maximum fluorescence value to be treated is calculated and used as the second fluorescence accumulation.

11. The method for evaluating nucleic acid test results according to claim 6, wherein, The determination of the first energy transfer value within the interval from the specified distance to the first distance between the donor and the recipient includes: Based on the transfer efficiency function of the FRET, the integral value of the distance between the donor and the receiver within the interval from the specified distance to the first distance is calculated as the first energy transfer value; The determination of the second energy transfer value within the interval between the second distance and the specified distance, which is the distance between the donor and the recipient, includes: Based on the transfer efficiency function of the FRET, the integral value of the distance between the donor and the recipient within the interval from the second distance to the specified distance is calculated as the second energy transfer value.

12. The method for evaluating nucleic acid detection results according to claim 6, wherein, The difference between the first distance and the specified distance is the specified distance difference value; The difference between the specified distance and the second distance is the specified distance difference value.

13. The method for evaluating nucleic acid detection results according to claim 12, wherein, The specified distance difference is a specified multiple of the specified distance.

14. The method for evaluating nucleic acid test results according to claim 13, wherein, The specified multiple is 0.

5.

15. A nucleic acid test result evaluation device, comprising: Memory; A processor, coupled to a memory, is configured to execute instructions stored in the memory to implement the nucleic acid detection result evaluation method as described in any one of claims 1-14.

16. A computer-readable storage medium, wherein, A computer-readable storage medium stores computer instructions that, when executed by a processor, implement the nucleic acid detection result evaluation method as described in any one of claims 1-14.

17. A computer program product comprising computer instructions, wherein the computer instructions, when executed by a processor, implement the nucleic acid detection result evaluation method as described in any one of claims 1-14.

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