A PCR instrument status detection and analysis method and system

Abstract

The present invention discloses a PCR instrument state detection and analysis method and system, which relates to the technical field of PCR instrument state detection, including defining the start and end marks in the PCR reaction stage by analyzing PCR instrument information and sample information, thereby identifying each PCR reaction stage in the PCR instrument processing process. The direct state index of the PCR instrument is determined according to the first reaction index of the PCR reaction stage, the transition process reaction stage and the single stage, and the indirect state index of the PCR instrument is determined according to the second reaction characteristic, so as to comprehensively analyze the PCR instrument state. The state index of the PCR instrument is determined by combining the direct state index and the indirect state index of the PCR instrument, and the direct state index and the indirect state index are combined to comprehensively and three-dimensionally analyze the state of the PCR instrument, thereby improving the accuracy and adaptability of the PCR instrument state detection and analysis, and ensuring the safe and stable operation of the PCR instrument.

Description

Technical Field

[0001] The present invention relates to the technical field of PCR instrument status detection, and in particular to a PCR instrument status detection and analysis method and system. Background Art

[0002] PCR instrument, full name Polymerase Chain Reaction Amplifier, is an instrument that uses PCR (Polymerase Chain Reaction) technology to amplify specific DNA fragments. PCR technology is a molecular biology technology that synthesizes DNA in vitro. Through the three basic steps of cyclic denaturation-annealing-extension, a specific DNA fragment can be amplified millions of times in a short period of time. PCR instruments have been widely used in medical and biological laboratories, such as to determine whether a specimen will show a map of a certain genetic disease, diagnosis of infectious diseases, gene replication, and paternity testing.

[0003] The PCR instrument status analysis program is based on the strict requirements of molecular biology experiments for high precision and high stability of PCR instruments. With the widespread application of gene amplification technology, PCR instruments, as core equipment, have a direct impact on the accuracy and reliability of experimental results. It aims to comprehensively evaluate key parameters such as temperature control and time control of PCR instruments by combining direct measurement with indirect analysis, to ensure that the instrument can operate stably and efficiently in gene amplification experiments, and to provide reliable technical support for scientific research and clinical diagnosis.

[0004] In the prior art, the detection and analysis of the status of a PCR instrument is often only judged based on the sample results after processing by the PCR instrument, without considering the relevant circumstances during the processing of the PCR instrument, resulting in low accuracy and poor adaptability of the detection and analysis of the status of the PCR instrument, and failing to ensure the safe and stable operation of the PCR instrument.

[0005] Therefore, how to improve the accuracy and adaptability of PCR instrument status detection and analysis is a technical problem that needs to be solved. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of low accuracy and poor adaptability of PCR instrument status detection and analysis in the prior art, and to propose a PCR instrument status detection and analysis method, which includes:

[0007] Acquire PCR instrument information and sample information, and define the start and end marks in the PCR reaction stage by analyzing the PCR instrument information and sample information, and identify each PCR reaction stage in the PCR instrument processing process according to the start and end marks in the PCR reaction stage, so as to obtain reaction data corresponding to each PCR reaction stage;

[0008] Extracting the first reaction feature according to the reaction data corresponding to the PCR reaction stage, determining the transition process reaction stage between the PCR reaction stages according to the processing time of the PCR instrument on the sample, allocating the first reaction index according to the PCR reaction stage and the transition process reaction stage, obtaining the first reaction index of the single stage, and determining the direct state index of the PCR instrument according to the PCR reaction stage, the transition process reaction stage and the first reaction index of the single stage;

[0009] Obtaining the processing result of the PCR instrument on the sample, analyzing the processing result of the PCR instrument on the sample to obtain result data, extracting the second reaction feature according to the result data, and determining the indirect state indicator of the PCR instrument according to the second reaction feature;

[0010] The state index of the PCR instrument is determined by combining the direct state index and the indirect state index of the PCR instrument, and the performance state of the PCR instrument is described by the state index of the PCR instrument.

[0011] In some embodiments of the present application, the start and end marks of the PCR reaction phase are defined by analyzing the PCR instrument information and the sample information, including:

[0012] Analyze each category of information in the PCR instrument information and sample information to determine the standard temperature range of the PCR reaction stage corresponding to each category of information, use the intersection range or temperature value of the standard temperature ranges of the PCR reaction stage of multiple categories of information as the start and end mark of the PCR reaction stage, set the standard duration corresponding to each PCR reaction stage by combining each category of information, and use the standard duration as the end mark of the PCR reaction stage.

[0013] In some embodiments of the present application, each PCR reaction stage in the PCR instrument processing process is identified according to the start and end marks in the PCR reaction stage, so as to obtain the reaction data corresponding to each PCR reaction stage, including:

[0014] Reaction data include instrument acquisition data and deviation data;

[0015] Each PCR reaction stage is identified by relying on the intersection range or temperature value of the standard temperature range, and the actual duration of each PCR reaction stage is determined. The instrument acquisition data of each PCR reaction stage is obtained, and the actual duration of each PCR reaction stage is compared with the standard duration to obtain the deviation between the two times, thereby generating deviation data.

[0016] In some embodiments of the present application, the transition process reaction stage between PCR reaction stages is determined by the processing time of the sample by the PCR instrument, and the first reaction index is allocated according to the PCR reaction stage and the transition process reaction stage, including:

[0017] A processing timeline is constructed through the processing time of the sample by the PCR instrument, and each PCR reaction stage in the identified PCR instrument processing process is marked on the corresponding time node of the processing timeline. The process processing timeline between adjacent PCR reaction stages on the processing timeline is used as a transition process reaction stage, and the first reaction index is allocated according to the corresponding time nodes of the PCR reaction stage and the transition process reaction stage on the processing timeline.

[0018] In some embodiments of the present application, direct status indicators of the PCR instrument are determined based on the PCR reaction stage, the transition process reaction stage, and the first reaction indicators of the single stage, including:

[0019] The first reaction index of a single stage includes a temperature index, a fluorescence signal index and a deviation index. The temperature index and the fluorescence signal index are obtained from the data collected by the instrument. The deviation index of the PCR reaction stage is obtained from the deviation data. The deviation index of the transformation process reaction stage is obtained from the process duration.

[0020] The transition process reaction stage is divided into an intra-cycle transition process reaction stage and an extra-cycle transition process reaction stage according to the PCR reaction stage adjacent to the transition process reaction stage, and the direct state index of the PCR instrument is determined according to the temperature index, fluorescence signal index and deviation index of each of the PCR reaction stage, the intra-cycle transition process reaction stage and the extra-cycle transition process reaction stage;

[0021] in, It is a direct indicator of the status of the PCR instrument. It is the status indicator of the PCR reaction stage. It is the state indicator of the reaction stage of the transformation process. , are the combined weights of the state indicators of the PCR reaction stage and the state indicators of the transformation process reaction stage, is the number of PCR reaction stages, For the The conversion factor corresponding to the type of PCR reaction stage is: , They are the weights corresponding to the temperature index and the fluorescence signal index in the PCR reaction stage. For the Temperature indicators for each PCR reaction stage, For the Fluorescence signal indicators of each PCR reaction stage, For the The deviation index size of each PCR reaction stage, are the first constant, the second constant and the third constant respectively, is the number of transformation reaction stages within the cycle, They are the weights corresponding to the temperature index and the fluorescence signal index in the reaction stage of the transformation process in the cycle, They are Temperature index and fluorescence signal index in the reaction stage of the transformation process within a cycle, It is The duration of the reaction phase of the transformation process, is the number of reaction stages of the transformation process outside the cycle, They are the weights corresponding to the temperature index and the fluorescence signal index in the reaction stage of the transformation process outside the cycle, and They are Temperature indicators and fluorescence signal indicators in the reaction stage of the transformation process outside the cycle.

[0022] In some embodiments of the present application, extracting the second reaction feature according to the result data includes:

[0023] The result data includes PCR amplification curve, Ct value and instrument acquisition data. A complete multiple PCR reaction stages are regarded as a single cycle. The transition process reaction stage within the cycle and the adjacent PCR reaction stage are recorded as the transition combination within the cycle. The transition process reaction stage outside the cycle and the adjacent PCR reaction stage are recorded as the transition combination outside the cycle. The changes of temperature index and fluorescence signal index in the transition combination within the cycle and the transition combination outside the cycle are calculated according to the instrument acquisition data, so as to integrate the changes under all cycle times and obtain the transition index.

[0024] Analyze the PCR amplification curve and identify different periods on the PCR amplification curve, determine the slope and special points according to the different periods of the PCR amplification curve, determine the Ct index according to the special points and the Ct value, and determine the amplification efficiency index according to the slope;

[0025] The transition index, Ct index and amplification efficiency index are used as the second reaction characteristics.

[0026] In some embodiments of the present application, the indirect status indicator of the PCR instrument is determined based on the second reaction characteristic, including:

[0027] Determine the indirect status index of the PCR instrument based on the transition index, Ct index and amplification efficiency index;

[0028] ;

[0029] in, It is an indirect status indicator of the PCR instrument. is the conversion coefficient corresponding to the transition indicator, is a transition indicator, is the conversion coefficient corresponding to the Ct indicator, is the Ct index, is the conversion coefficient corresponding to the amplification efficiency index, It is an indicator of amplification efficiency.

[0030] In some embodiments of the present application, the state indicator of the PCR instrument is determined by combining the direct state indicator and the indirect state indicator of the PCR instrument, including:

[0031] The direct status index and the indirect status index of the PCR instrument are weighted and summed by preset weights to obtain the status index of the PCR instrument.

[0032] Correspondingly, the present application also provides a PCR instrument status detection and analysis system, including:

[0033] An identification module is used to obtain PCR instrument information and sample information, and define the start and end marks in the PCR reaction stage by analyzing the PCR instrument information and sample information, and identify each PCR reaction stage in the PCR instrument processing process according to the start and end marks in the PCR reaction stage, so as to obtain reaction data corresponding to each PCR reaction stage;

[0034] A first determination module is used to extract a first reaction feature according to reaction data corresponding to a PCR reaction stage, determine a transition process reaction stage between PCR reaction stages according to a processing time of the sample by the PCR instrument, allocate a first reaction index according to the PCR reaction stage and the transition process reaction stage, obtain a first reaction index of a single stage, and determine a direct state index of the PCR instrument according to the PCR reaction stage, the transition process reaction stage and the first reaction index of the single stage;

[0035] A second determination module is used to obtain the processing result of the PCR instrument on the sample, analyze the processing result of the PCR instrument on the sample to obtain result data, extract the second reaction feature according to the result data, and determine the indirect state indicator of the PCR instrument according to the second reaction feature;

[0036] The analysis module is used to determine the status indicator of the PCR instrument by combining the direct status indicator and the indirect status indicator of the PCR instrument, and to describe the performance status of the PCR instrument through the status indicator of the PCR instrument.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. Define the start and end marks of the PCR reaction stage by analyzing the PCR instrument information and sample information, so as to identify each PCR reaction stage in the PCR instrument processing process, and lay a solid foundation for the determination of subsequent direct status indicators and indirect status indicators.

[0039] 2. The direct status indicators of the PCR instrument are determined based on the first reaction indicators of the PCR reaction stage, the transition process reaction stage and the single stage, and the indirect status indicators of the PCR instrument are determined based on the second reaction characteristics. The direct status indicators during the PCR instrument processing process and the indirect status indicators of the results after the PCR instrument processing are considered, so as to comprehensively analyze the PCR instrument status.

[0040] 3. Combine the direct status indicators and indirect status indicators of the PCR instrument to determine the status indicators of the PCR instrument. Combining the direct status indicators with the indirect status indicators, the status of the PCR instrument is comprehensively and three-dimensionally analyzed, the accuracy and adaptability of the PCR instrument status detection and analysis are improved, and the safe and stable operation of the PCR instrument is ensured. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A schematic diagram of a process for detecting and analyzing the state of a PCR instrument proposed by the present invention;

[0042] Figure 2 This is a structural schematic diagram of a PCR instrument status detection and analysis system proposed by the present invention. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0044] Reference Figure 1 , a PCR instrument status detection and analysis method, comprising the following steps:

[0045] Step S101, obtaining PCR instrument information and sample information, and defining the start and end marks in the PCR reaction stage by analyzing the PCR instrument information and sample information, and identifying each PCR reaction stage in the PCR instrument processing process according to the start and end marks in the PCR reaction stage, thereby obtaining reaction data corresponding to each PCR reaction stage.

[0046] In this embodiment, the PCR instrument information includes the instrument model and manufacturer, the accuracy and stability of the temperature control system, the programmable temperature and time setting range, and the heating and cooling rates. The sample information includes the sample type (such as DNA, RNA), the length and GC content of the target sequence, the primer design and concentration, the concentration and purity of the template DNA, the required enzyme and buffer type, etc. For example, the complexity of the template DNA in different samples is different, which will affect the difficulty of dissociating the double-stranded DNA into single strands in the denaturation step. DNA with high complexity may require a higher denaturation temperature and a longer denaturation time.

[0047] PCR instruments achieve rapid, specific, and highly sensitive amplification of DNA by precisely controlling temperature. A heating block or heating core is usually used inside the instrument to maintain a constant temperature, and a temperature sensor is used for precise temperature measurement. During the PCR reaction, the instrument controls the temperature through cycles so that the DNA template and primers can obtain optimal temperature conditions during the denaturation, annealing, and extension stages. Specifically, the PCR reaction stages include the following, which are repeated multiple times to achieve the amplification of a specific DNA fragment by millions of times in a short period of time.

[0048] Denaturation: At high temperatures (usually 95°C), double-stranded DNA is unwound into single strands.

[0049] Annealing: The temperature drops to about 55°C, and the primers pair up with the complementary sequences of the template DNA single strand.

[0050] Extension: Under the action of DNA polymerase (such as Taq enzyme), dNTP is used as the reaction raw material, and according to the principles of base pairing and semiconservative replication, a new semiconservative replication chain complementary to the template DNA chain is synthesized.

[0051] The start and end marks of the PCR reaction phase. Here the mark refers to the temperature. The following is a specific example:

[0052] Transformation stage:

[0053] Start mark: The temperature reaches the preset denaturation temperature, at which time the DNA double strands begin to separate.

[0054] End mark: The temperature begins to drop, marking the end of the denaturation phase and preparing to enter the annealing phase.

[0055] Annealing stage:

[0056] Start mark: The temperature drops to the preset annealing temperature, at which time the primer begins to bind to the template DNA.

[0057] End mark: The temperature begins to rise, marking the end of the annealing stage and preparing to enter the extension stage.

[0058] Extension phase:

[0059] Start mark: The temperature reaches the preset extension temperature, at which time the DNA polymerase begins to synthesize a new DNA chain.

[0060] End mark: The temperature begins to change (such as starting to drop or rise to enter the next cycle), marking the end of the extension phase.

[0061] The denaturation-annealing-extension phase is a single complete cycle, and these three phases are repeated, usually for 20-40 cycles (the specific number of cycles depends on the experimental requirements and the concentration of the target DNA). Each cycle doubles the amount of target DNA (ideally), thereby achieving rapid DNA amplification. Ultimately, through multiple cycles of denaturation-annealing-extension, the PCR instrument is able to process the sample and produce a sufficient amount of target DNA. The process between the denaturation-annealing and annealing-extension phases belongs to the transition process reaction phase (the transition process reaction phase within the cycle), and the process between the extension-denaturation phase of the next cycle also belongs to the transition process reaction phase (the transition process reaction phase outside the cycle).

[0062] In some embodiments of the present application, the start and end marks of the PCR reaction phase are defined by analyzing the PCR instrument information and the sample information, including:

[0063] Analyze each category of information in the PCR instrument information and sample information to determine the standard temperature range of the PCR reaction stage corresponding to each category of information, use the intersection range or temperature value of the standard temperature ranges of the PCR reaction stage of multiple categories of information as the start and end mark of the PCR reaction stage, set the standard duration corresponding to each PCR reaction stage by combining each category of information, and use the standard duration as the end mark of the PCR reaction stage.

[0064] In this embodiment, the preset temperature value and the standard duration of each reaction stage are set by comprehensively considering the sample information and the PCR instrument information.

[0065] In some embodiments of the present application, each PCR reaction stage in the PCR instrument processing process is identified according to the start and end marks in the PCR reaction stage, so as to obtain the reaction data corresponding to each PCR reaction stage, including:

[0066] Reaction data include instrument acquisition data and deviation data;

[0067] Each PCR reaction stage is identified by relying on the intersection range or temperature value of the standard temperature range, and the actual duration of each PCR reaction stage is determined. The instrument acquisition data of each PCR reaction stage is obtained, and the actual duration of each PCR reaction stage is compared with the standard duration to obtain the deviation between the two times, thereby generating deviation data.

[0068] In this embodiment, the instrument collected data is the relevant data recorded by the PCR instrument, such as temperature, temperature fluctuation, fluorescence signal intensity, etc., and the deviation data is the deviation time. The larger the deviation time, the greater the possibility of reaction abnormality.

[0069] Step S102, extracting the first reaction feature according to the reaction data corresponding to the PCR reaction stage, determining the transition process reaction stage between the PCR reaction stages through the processing time of the sample by the PCR instrument, allocating the first reaction index according to the PCR reaction stage and the transition process reaction stage, obtaining the first reaction index of the single stage, and determining the direct state index of the PCR instrument according to the PCR reaction stage, the transition process reaction stage and the first reaction index of the single stage.

[0070] In this embodiment, extracting the first reaction feature is to screen out the relevant data, which mainly refers to the data related to the temperature control performance and the fluorescence detection performance. The PCR reaction stage and the reaction stage of the transformation process are determined according to the complete processing time of the PCR instrument for the sample. And the first reaction index is allocated according to the reaction stage. The temperature control performance of the PCR instrument directly affects the success or failure of the PCR experiment. Parameters such as the heating rate, the cooling rate, the temperature fluctuation, the temperature indication error and the temperature uniformity are important indicators for measuring the performance of the instrument. The abnormality of these parameters may lead to the instability of the PCR reaction conditions, thereby affecting the experimental results. Fluorescence detection performance is one of the core performances of the real-time fluorescence quantitative PCR instrument. Parameters such as fluorescence intensity repeatability, fluorescence intensity uniformity and fluorescence intensity linearity reflect the stability and accuracy of the instrument in fluorescence signal detection. The abnormality of these parameters may cause the experimental results to be inaccurate or unable to be analyzed. Therefore, temperature and fluorescence are two directions that can directly reflect the performance state of PCR.

[0071] In some embodiments of the present application, the transition process reaction stage between PCR reaction stages is determined by the processing time of the sample by the PCR instrument, and the first reaction index is allocated according to the PCR reaction stage and the transition process reaction stage, including:

[0072] A processing timeline is constructed through the processing time of the sample by the PCR instrument, and each PCR reaction stage in the identified PCR instrument processing process is marked on the corresponding time node of the processing timeline. The process processing timeline between adjacent PCR reaction stages on the processing timeline is used as a transition process reaction stage, and the first reaction index is allocated according to the corresponding time nodes of the PCR reaction stage and the transition process reaction stage on the processing timeline.

[0073] In this embodiment, the main purpose here is to determine the time nodes of both the PCR reaction phase and the transformation process reaction phase.

[0074] In some embodiments of the present application, direct status indicators of the PCR instrument are determined based on the PCR reaction stage, the transition process reaction stage, and the first reaction indicators of the single stage, including:

[0075] The first reaction index of a single stage includes a temperature index, a fluorescence signal index and a deviation index. The temperature index and the fluorescence signal index are obtained from the data collected by the instrument. The deviation index of the PCR reaction stage is obtained from the deviation data. The deviation index of the transformation process reaction stage is obtained from the process duration.

[0076] The transition process reaction stage is divided into an intra-cycle transition process reaction stage and an extra-cycle transition process reaction stage according to the PCR reaction stage adjacent to the transition process reaction stage, and the direct state index of the PCR instrument is determined according to the temperature index, fluorescence signal index and deviation index of each of the PCR reaction stage, the intra-cycle transition process reaction stage and the extra-cycle transition process reaction stage;

[0077] ;

[0078] in, It is a direct indicator of the status of the PCR instrument. It is the status indicator of the PCR reaction stage. It is the state indicator of the reaction stage of the transformation process. , are the combined weights of the state indicators of the PCR reaction stage and the state indicators of the transformation process reaction stage, is the number of PCR reaction stages, For the The conversion factor corresponding to the type of PCR reaction stage is: , They are the weights corresponding to the temperature index and the fluorescence signal index in the PCR reaction stage. For the Temperature indicators for each PCR reaction stage, For the Fluorescence signal indicators of each PCR reaction stage, For the The deviation index size of each PCR reaction stage, are the first constant, the second constant and the third constant respectively, is the number of transformation reaction stages within the cycle, They are the weights corresponding to the temperature index and the fluorescence signal index in the reaction stage of the transformation process in the cycle, They are Temperature index and fluorescence signal index in the reaction stage of the transformation process within a cycle, It is The duration of the reaction phase of the transformation process, is the number of reaction stages of the transformation process outside the cycle, They are the weights corresponding to the temperature index and the fluorescence signal index in the reaction stage of the transformation process outside the cycle, and They are Temperature indicators and fluorescence signal indicators in the reaction stage of the transformation process outside the cycle.

[0079] In this embodiment, the temperature index is an index that directly describes the quality of the temperature of the PCR instrument and is determined by comprehensively analyzing relevant temperature parameters, and the fluorescence signal index is an index that directly describes the quality of the fluorescence of the PCR instrument and is determined by comprehensively analyzing relevant fluorescence parameters. The deviation index of the PCR reaction stage is obtained from the deviation data, and the deviation index of the transition process reaction stage is obtained from the process duration. Here, the completion efficiency of the transition process reaction stage is determined from the side by time. It is divided into the transition process reaction stage within the cycle and the transition process reaction stage outside the cycle. The process between the denaturation-annealing and annealing-extension stages belongs to the transition process reaction stage (the transition process reaction stage within the cycle), and the process between the extension-denaturation stage of the next cycle also belongs to the transition process reaction stage (the transition process reaction stage outside the cycle).

[0080] In this embodiment, For the The conversion coefficients corresponding to the types of PCR reaction stages (denaturation, annealing, extension). The PCR reaction stages are integrated. It represents the sum of the influence of temperature and fluorescence index corrected by the deviation index. The reaction phase of the transformation process within the cycle and the reaction phase of the transformation process outside the cycle are integrated. It represents the correction of the transformation time (efficiency) to the sum of the effects of temperature and fluorescence index.

[0081] Step S103, obtaining the processing result of the PCR instrument on the sample, analyzing the processing result of the PCR instrument on the sample to obtain result data, extracting the second reaction feature according to the result data, and determining the indirect state indicator of the PCR instrument according to the second reaction feature.

[0082] In this embodiment, the result data after the reaction is completed, such as fluorescence intensity, amplification curve, etc., are read or derived from the PCR instrument.

[0083] In some embodiments of the present application, extracting the second reaction feature according to the result data includes:

[0084] The result data includes PCR amplification curve, Ct value and instrument acquisition data. A complete multiple PCR reaction stages are regarded as a single cycle. The transition process reaction stage within the cycle and the adjacent PCR reaction stage are recorded as the transition combination within the cycle. The transition process reaction stage outside the cycle and the adjacent PCR reaction stage are recorded as the transition combination outside the cycle. The changes of temperature index and fluorescence signal index in the transition combination within the cycle and the transition combination outside the cycle are calculated according to the instrument acquisition data, so as to integrate the changes under all cycle times and obtain the transition index.

[0085] Analyze the PCR amplification curve and identify different periods on the PCR amplification curve, determine the slope and special points according to the different periods of the PCR amplification curve, determine the Ct index according to the special points and the Ct value, and determine the amplification efficiency index according to the slope;

[0086] The transition index, Ct index and amplification efficiency index are used as the second reaction characteristics.

[0087] In this embodiment, the PCR amplification curve is a curve that describes the dynamic process of PCR, usually an S-shaped curve. During the PCR process, as the number of cycles increases, DNA polymerase catalyzes the extension of the DNA chain, the amount of amplified product gradually increases, and the corresponding fluorescent signal continues to increase. The abscissa of the amplification curve represents the number of cycles of amplification (Cycle), and the ordinate represents the intensity of fluorescence (Fluorescence). The Ct value (Cycle Threshold) refers to the number of cycles corresponding to when the fluorescent signal of the amplified product reaches the set fluorescence threshold (Fluorescence Threshold) during the PCR amplification process. It can also be understood as the number of cycles corresponding to when the initial template is amplified to a certain product amount value during the qPCR experiment. The Ct value is an important parameter in fluorescent quantitative PCR experiments, which is used to calculate the results of gene expression differences or gene copy numbers.

[0088] In the present embodiment, the transition combination in the cycle is denaturation-transition process reaction stage-annealing, annealing-transition process reaction stage-extension, and the transition combination outside the cycle is extension-transition process reaction stage-next cycle. According to the temperature index and fluorescent signal index in the three processes in the combination, the change is obtained, and the transition combination under all cycle times is counted to generate a transition index, which is used to describe the change during the transition of the PCR reaction stage. Analyze the PCR amplification curve and identify the different periods on the PCR amplification curve, which is usually presented as an S-shaped curve, which contains three stages of the PCR reaction: exponential growth period, linear growth period and plateau period. Use professional software or algorithms to fit the curve and distinguish each period according to the slope.

[0089] In this embodiment, the slope and special points are determined according to different periods of the PCR amplification curve. The slope here refers to the slope of the exponential growth period. In the exponential growth stage, the slope of the curve can reflect the amplification efficiency because it grows fastest during this period. Special points include starting point, inflection point and end point. The starting point usually corresponds to the lowest point of the PCR amplification curve, which reflects the amount of the initial template DNA. The inflection point is the point where the slope on the amplification curve changes significantly, which usually corresponds to the moment when the amplification rate begins to slow down. The end point is the point when the amplification curve reaches the plateau phase, which reflects the amount of the final product DNA. The Ct index describes the rationality of Ct. No Ct value or abnormal Ct value indicates that the PCR instrument may be abnormal. The rationality of Ct is judged by combining the starting point, inflection point, end point and Ct value. The starting point corresponds to the lowest point of the PCR amplification curve, which reflects the amount of the initial template DNA. The height of the starting point directly affects the starting speed of the amplification reaction and the number of cycles required to reach the detectable level. If the starting point is higher, it means that the initial template amount is larger, and the amplification reaction may reach the plateau phase faster, and the Ct value may be relatively small at this time. On the contrary, a lower starting point indicates that the initial template amount is small, and the amplification reaction may require more cycles to reach a detectable level, so the Ct value may be relatively large. The inflection point is the point where the slope on the amplification curve changes significantly, which usually corresponds to the moment when the amplification rate begins to slow down. The position of the inflection point can reflect the efficiency of the amplification and certain limiting factors in the reaction system, such as enzyme activity, substrate concentration, etc. If the inflection point appears early, it may mean that the amplification reaction encounters limitations in the early stage, resulting in a slowdown in the amplification rate, which may affect the size of the Ct value. On the contrary, if the inflection point appears later, it means that the amplification reaction maintains a higher speed for a longer time, which may result in a relatively large Ct value.

[0090] In some embodiments of the present application, the indirect status indicator of the PCR instrument is determined based on the second reaction characteristic, including:

[0091] Determine the indirect status index of the PCR instrument based on the transition index, Ct index and amplification efficiency index;

[0092] ;

[0093] in, It is an indirect status indicator of the PCR instrument. is the conversion coefficient corresponding to the transition indicator, is a transition indicator, is the conversion coefficient corresponding to the Ct indicator, is the Ct index, is the conversion coefficient corresponding to the amplification efficiency index, It is an indicator of amplification efficiency.

[0094] Step S104, determining the status indicator of the PCR instrument by combining the direct status indicator and the indirect status indicator of the PCR instrument, and describing the performance status of the PCR instrument by the status indicator of the PCR instrument.

[0095] In some embodiments of the present application, the state indicator of the PCR instrument is determined by combining the direct state indicator and the indirect state indicator of the PCR instrument, including:

[0096] The direct status index and the indirect status index of the PCR instrument are weighted and summed by preset weights to obtain the status index of the PCR instrument.

[0097] Compared with the prior art, the present invention has the following beneficial effects:

[0098] 1. Define the start and end marks of the PCR reaction stage by analyzing the PCR instrument information and sample information, so as to identify each PCR reaction stage in the PCR instrument processing process, and lay a solid foundation for the determination of subsequent direct status indicators and indirect status indicators.

[0099] 2. The direct status indicators of the PCR instrument are determined based on the first reaction indicators of the PCR reaction stage, the transition process reaction stage and the single stage, and the indirect status indicators of the PCR instrument are determined based on the second reaction characteristics. The direct status indicators during the PCR instrument processing process and the indirect status indicators of the results after the PCR instrument processing are considered, so as to comprehensively analyze the PCR instrument status.

[0100] 3. Combine the direct status indicators and indirect status indicators of the PCR instrument to determine the status indicators of the PCR instrument. Combining the direct status indicators with the indirect status indicators, the status of the PCR instrument is comprehensively and three-dimensionally analyzed, the accuracy and adaptability of the PCR instrument status detection and analysis are improved, and the safe and stable operation of the PCR instrument is ensured.

[0101] Through the description of the above implementation methods, those skilled in the art can clearly understand that the present invention can be implemented by hardware, or by software plus a necessary general hardware platform. Based on such an understanding, the technical solution of the present invention can be embodied in the form of a software product, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including a number of instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in each implementation scenario of the present invention.

[0102] Correspondingly, the present application also provides a PCR instrument status detection and analysis system, such as Figure 2 As shown, including,

[0103] An identification module is used to obtain PCR instrument information and sample information, and define the start and end marks in the PCR reaction stage by analyzing the PCR instrument information and sample information, and identify each PCR reaction stage in the PCR instrument processing process according to the start and end marks in the PCR reaction stage, so as to obtain reaction data corresponding to each PCR reaction stage;

[0104] A first determination module is used to extract a first reaction feature according to reaction data corresponding to a PCR reaction stage, determine a transition process reaction stage between PCR reaction stages according to a processing time of the sample by the PCR instrument, allocate a first reaction index according to the PCR reaction stage and the transition process reaction stage, obtain a first reaction index of a single stage, and determine a direct state index of the PCR instrument according to the PCR reaction stage, the transition process reaction stage and the first reaction index of the single stage;

[0105] A second determination module is used to obtain the processing result of the PCR instrument on the sample, analyze the processing result of the PCR instrument on the sample to obtain result data, extract the second reaction feature according to the result data, and determine the indirect state indicator of the PCR instrument according to the second reaction feature;

[0106] The analysis module is used to determine the status indicator of the PCR instrument by combining the direct status indicator and the indirect status indicator of the PCR instrument, and to describe the performance status of the PCR instrument through the status indicator of the PCR instrument.

[0107] Those skilled in the art will appreciate that the accompanying drawings are merely schematic diagrams of a preferred implementation scenario, and the modules or processes in the accompanying drawings are not necessarily required for implementing the present invention.

[0108] Those skilled in the art will appreciate that the modules in the system in the implementation scenario can be distributed in the system of the implementation scenario according to the implementation scenario description, or can be changed accordingly and located in one or more systems different from the implementation scenario. The modules in the above implementation scenario can be combined into one module, or can be further split into multiple submodules.

[0109] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A PCR instrument status detection and analysis method, characterized in that: include, Acquire PCR instrument information and sample information, and define the start and end marks of the PCR reaction phase by analyzing the PCR instrument information and sample information, including: Analyze each category of information in the PCR instrument information and the sample information to determine the standard temperature range of the PCR reaction stage corresponding to each category of information, use the intersection range or temperature value of the standard temperature ranges of the PCR reaction stage of multiple categories of information as the start and end mark of the PCR reaction stage, set the standard duration corresponding to each PCR reaction stage by combining each category of information, and use the standard duration as the end mark of the PCR reaction stage; According to the start and end marks of the PCR reaction stage, each PCR reaction stage in the PCR instrument processing process is identified, so as to obtain the reaction data corresponding to each PCR reaction stage, including: Reaction data include instrument acquisition data and deviation data; Relying on the intersection range or temperature value of the standard temperature range to identify each PCR reaction stage, and determine the actual duration of each PCR reaction stage, obtain the instrument acquisition data of each PCR reaction stage, and compare the actual duration of each PCR reaction stage with the standard duration to obtain the deviation of the two times, thereby generating deviation data, and extracting the first reaction feature according to the reaction data corresponding to the PCR reaction stage; The transition process reaction stage between the PCR reaction stages is determined by the processing time of the sample by the PCR instrument, and the first reaction index is allocated according to the PCR reaction stage and the transition process reaction stage to obtain the first reaction index of the single stage, including: A processing time axis is constructed according to the processing time of the sample by the PCR instrument, each PCR reaction stage in the processing process of the identified PCR instrument is marked on the corresponding time node of the processing time axis, and the process processing time axis between adjacent PCR reaction stages on the processing time axis is used as a transformation process reaction stage, and the first reaction index is allocated according to the corresponding time nodes of the PCR reaction stage and the transformation process reaction stage on the processing time axis; The direct status indicators of the PCR instrument are determined based on the PCR reaction stage, the transition process reaction stage and the first reaction indicators of the single stage, including: The first reaction index of a single stage includes a temperature index, a fluorescence signal index and a deviation index. The temperature index and the fluorescence signal index are obtained from the data collected by the instrument. The deviation index of the PCR reaction stage is obtained from the deviation data. The deviation index of the transformation process reaction stage is obtained from the process duration. The completion efficiency of the transformation process reaction stage is determined from the side through time. The transition process reaction stage is divided into an intra-cycle transition process reaction stage and an extra-cycle transition process reaction stage according to the PCR reaction stage adjacent to the transition process reaction stage, and the direct state index of the PCR instrument is determined according to the temperature index, fluorescence signal index and deviation index of each of the PCR reaction stage, the intra-cycle transition process reaction stage and the extra-cycle transition process reaction stage; ； in, It is a direct indicator of the status of the PCR instrument. It is the status indicator of the PCR reaction stage. It is the state indicator of the reaction stage of the transformation process. , are the combined weights of the state indicators of the PCR reaction stage and the state indicators of the transformation process reaction stage, is the number of PCR reaction stages, For the The conversion factor corresponding to the type of PCR reaction stage is: , They are the weights corresponding to the temperature index and the fluorescence signal index in the PCR reaction stage. For the Temperature indicators for each PCR reaction stage, For the Fluorescence signal indicators of each PCR reaction stage, For the The deviation index size of each PCR reaction stage, are the first constant, the second constant and the third constant respectively, is the number of transformation reaction stages within the cycle, , They are the weights corresponding to the temperature index and the fluorescence signal index in the reaction stage of the transformation process in the cycle, , They are Temperature index and fluorescence signal index in the reaction stage of the transformation process within a cycle, It is The duration of the reaction phase of the transformation process, is the number of reaction stages of the transformation process outside the cycle, , They are the weights corresponding to the temperature index and the fluorescence signal index in the reaction stage of the transformation process outside the cycle, and They are Temperature index and fluorescence signal index in the reaction stage of the transformation process outside the cycle; Obtaining the processing result of the PCR instrument on the sample, analyzing the processing result of the PCR instrument on the sample to obtain result data, and extracting the second reaction feature according to the result data, including, The result data includes PCR amplification curve, Ct value and instrument acquisition data. A complete multiple PCR reaction stages are regarded as a single cycle. The transition process reaction stage within the cycle and the adjacent PCR reaction stage are recorded as the transition combination within the cycle. The transition process reaction stage outside the cycle and the adjacent PCR reaction stage are recorded as the transition combination outside the cycle. The changes of temperature index and fluorescence signal index in the transition combination within the cycle and the transition combination outside the cycle are calculated according to the instrument acquisition data, so as to integrate the changes under all cycle times and obtain the transition index. Analyze the PCR amplification curve and identify different periods on the PCR amplification curve, determine the slope and special points according to the different periods of the PCR amplification curve, determine the Ct index according to the special points and the Ct value, and determine the amplification efficiency index according to the slope; The transition index, Ct index and amplification efficiency index are used as the second reaction characteristics; And based on the second reaction characteristics, the indirect status indicators of the PCR instrument are determined, including, Determine the indirect status index of the PCR instrument based on the transition index, Ct index and amplification efficiency index; ； in, It is an indirect status indicator of the PCR instrument. is the conversion coefficient corresponding to the transition indicator, is a transition indicator, is the conversion coefficient corresponding to the Ct indicator, is the Ct index, is the conversion coefficient corresponding to the amplification efficiency index, It is an indicator of amplification efficiency; The state indicators of the PCR instrument are determined by combining the direct state indicators and indirect state indicators of the PCR instrument, including: The direct status index and the indirect status index of the PCR instrument are weighted and summed by preset weights to obtain the status index of the PCR instrument.

2. A PCR instrument status detection and analysis system, used to implement a PCR instrument status detection and analysis method as claimed in claim 1, characterized in that: include, An identification module is used to obtain PCR instrument information and sample information, and define the start and end marks in the PCR reaction stage by analyzing the PCR instrument information and sample information, and identify each PCR reaction stage in the PCR instrument processing process according to the start and end marks in the PCR reaction stage, so as to obtain reaction data corresponding to each PCR reaction stage; A first determination module is used to extract a first reaction feature according to reaction data corresponding to a PCR reaction stage, determine a transition process reaction stage between PCR reaction stages according to a processing time of the sample by the PCR instrument, allocate a first reaction index according to the PCR reaction stage and the transition process reaction stage, obtain a first reaction index of a single stage, and determine a direct state index of the PCR instrument according to the PCR reaction stage, the transition process reaction stage and the first reaction index of the single stage; A second determination module is used to obtain the processing result of the PCR instrument on the sample, analyze the processing result of the PCR instrument on the sample to obtain result data, extract the second reaction feature according to the result data, and determine the indirect state indicator of the PCR instrument according to the second reaction feature; The analysis module is used to determine the status indicator of the PCR instrument by combining the direct status indicator and the indirect status indicator of the PCR instrument, and to describe the performance status of the PCR instrument through the status indicator of the PCR instrument.

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