Method and device for classifying amplification containers

Abstract

The invention relates to a method and a device for classifying amplification containers. The method comprises the following steps: for each of a plurality of amplification containers, respectively carrying out signal acquisition at a plurality of given time points so as to obtain an original real-time signal data set; based on the original real-time signal data set, carrying out primary classification on at least one of the plurality of amplification containers so as to obtain a primarily judged amplification container and a primarily judged non-amplification container; determining an amplification baseline based on at least one part of signals of the primarily judged non-amplification container, and correcting at least one part of the original real-time signal data set by adopting the amplification baseline so as to obtain a corrected real-time signal data set; and based on the corrected real-time signal data set, carrying out secondary classification on at least one of the plurality of amplification containers so as to obtain a final judgment amplification container and a final judgment non-amplification container. The classification method enables result data of different amplification systems, instrument platforms, laboratories and the like to have comparability.

Description

Technical Field

[0001] The present application relates to the field of bioinformatics, and in particular, to a method for classifying amplification containers. Background Art

[0002] In the field of biological sample analysis and molecular diagnostic technology, in order to ensure that the detection method has sufficient sensitivity and specificity, the amplification of the target gene or nucleotide fragment is a commonly used technical means. Polymerase chain reaction (PCR) is the most commonly used method for rapid and large-scale replication of target nucleic acid molecules in vitro or in a test tube. It can be used to amplify specific DNA fragments and realize qualitative and quantitative detection of biochemical analytes such as nucleic acids. PCR uses variable temperature cycles to allow the double-stranded DNA used as a template to complete high-temperature denaturation, primer annealing, and complementary chain extension synthesis in sequence under the enzymatic action of polymerase. A complete variable temperature cycle includes the above three steps. In theory, it can double the number of templates. Continuously repeating the variable temperature cycle can generate exponentially growing PCR specific end products. The number of variable temperature cycles determines the amplification multiple of the initial template. Usually, after 30 to 40 variable temperature cycles, the target gene or nucleotide fragment can be amplified by about 10 9 times, thus reaching a level that can be detected by the instrument sensor.

[0003] Nucleic acid isothermal amplification technology is a new type of solution proposed to overcome the various limitations and shortcomings in the application of PCR technology. They can quickly expand the number of copies of target DNA or RNA fragments at a constant temperature. This type of technology can reduce the complicated steps of sample and reagent preparation, completely get rid of the dependence on thermal cyclers, and can greatly simplify the complexity of nucleic acid detection schemes, which is of great significance for the application of on-site instant detection. Common isothermal amplification techniques include loop-mediated amplification (LAMP), rolling circle amplification (RCA), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), single primer isothermal amplification (SPIA) and helicase dependent amplification (HDA).

[0004] These isothermal amplification techniques do not need to rely on variable temperature cycles to change the conformation of the template chain. Most of them use a polymerase-mediated chain displacement mechanism that is different from thermal denaturation in PCR to achieve the release of the template chain and the annealing and binding of the primers. The polymerase-mediated nucleic acid chain displacement reaction is a technology widely used in nucleic acid amplification reactions, and it is also a simulation and variant reaction of the nucleic acid replication process in organisms. The process mainly includes two processes: primer extension under the action of a polymerase with chain displacement activity and the new nucleic acid chain generated by the extension replaces the original nucleic acid chain downstream. The polymerase-mediated chain displacement leads to the unwinding of the original double strand and the generation of a new double strand, resulting in an amplification reaction. This displacement reaction replaces the high-temperature unwinding process in the traditional variable temperature reaction, can be carried out in a wide temperature range, and is simple and convenient to operate. In recent years, the nucleic acid chain displacement reaction has been widely used in various fields of molecular biology due to its high specificity and high sensitivity detection characteristics, and has also received great attention in the amplification of detection signals and diagnostic biosensor detection.

[0005] Based on the above principles, by designing and constructing templates, primers or intermediates with specific structures, the cyclic reuse of templates and intermediates or the parallel extension of primers can be achieved. This self-circulating chain displacement amplification mechanism often produces multiple products through repeated amplification of a template to achieve faster product synthesis, which can greatly improve the efficiency and sensitivity of amplification. Self-circulating chain displacement amplification includes LAMP, RCA, MDA, SDA, SPIA and HDA technologies. Among them, the most widely used LAMP technology has the typical characteristics and advantages of self-circulating chain displacement amplification, and the amplification efficiency and sensitivity are also the best.

[0006] Therefore, there is an urgent need in the art to develop a universal isothermal amplification real-time signal result analysis method. Summary of the invention

[0007] This application is filed by the inventor based on the discovery of the following problems and facts:

[0008] Current isothermal amplification real-time detection technology has the following limitations:

[0009] (1) The real-time detection temperature control and signal acquisition procedures adopted by various technical solutions based on different labeling methods are different, resulting in differences in the collected signals, and the real-time detection results cannot be compared in parallel;

[0010] (2) The real-time detection procedures of various technical solutions based on the same labeling method are artificially preset, and the preset reading number and reading time interval are relatively arbitrary, lacking a unified standard, resulting in the total detection time being arbitrary and usually long. There are limitations in the detection procedures and result judgment standards for samples of different quantities, qualities, and types, such as strong positive, medium positive, weak positive, and negative;

[0011] (3) There is no unified standard for the calculation method of the quantitative index values ​​used in some technical solutions, and they are usually processed based on the default analysis method of the real-time PCR supporting software program (the algorithm is based on the PCR exponential kinetic model), which is not necessarily compatible and applicable to isothermal amplification (super-exponential kinetic model);

[0012] (4) Usually, the test results can only be qualitatively judged by the shape of the real-time amplification curve. There is a lack of a universal quantifiable indicator similar to the real-time PCR median to characterize the occurrence of positive signals, and it is impossible to achieve semi-quantitative or quantitative comparison of the test results;

[0013] (5) Unable to meet the automation and throughput requirements of nucleic acid testing.

[0014] Therefore, the present application satisfies the standardization, automation and flux requirements of analyzing and detecting a large number of nucleic acid sample scenarios by providing a method for analyzing real-time signal results of isothermal amplification. And this method is not limited to a specific amplification reaction kinetic model, but can be widely used in real-time detection schemes of various sequence-specific and non-specific markers, and nucleic acid detection results (such as single PCR tubes, 8-row tubes, 96 or 384-hole PCR plates, droplets and microfluidic amplification pools or other amplification containers) can be obtained more accurately and quickly, so that isothermal amplification can be similar to the quantitative index TTP (Time-to-positive) value (or TTD value, Time-to-detect) of the real-time PCR median, that is, the time required for the real-time fluorescent signal proportional to the accumulation concentration of the amplification (by) product to reach the positive signal threshold set (reporting positive time or detection time), to complete the evaluation parameters such as sensitivity, specificity, precision, repeatability, reproducibility, and detection limit, so that the independent detection results across amplification systems, across instrument platforms, and across laboratories have intra-batch and inter-batch result data comparability.

[0015] The present invention aims to solve one of the above technical problems to at least some extent or at least provide a useful commercial choice.

[0016] In the first aspect of the present invention, the present invention proposes a method for classifying amplification containers. According to an embodiment of the present invention, the method includes: for each of a plurality of amplification containers, signal acquisition is performed at a plurality of given time points respectively, so as to obtain an original real-time signal data set; based on the original real-time signal data set, a classification is performed for at least one of the plurality of amplification containers, so as to obtain a preliminary judgment amplification container and a preliminary judgment non-amplification container; based on at least a portion of the signal of the preliminary judgment non-amplification container, an amplification baseline is determined, and at least a portion of the original real-time signal data set is corrected using the amplification baseline, so as to obtain a corrected real-time signal data set; based on the corrected real-time signal data set, a secondary classification is performed for at least one of the plurality of amplification containers, so as to obtain a final judgment amplification container and a final judgment non-amplification container. According to an embodiment of the present invention, the classification method combines technical means such as data acquisition, real-time, automated processing and fine correction at multiple time points, and has a high degree of accuracy, real-time and automation. And the method is not limited to a specific amplification reaction kinetic model, and can be applied to sequence-specific and non-specific real-time labeling detection schemes, and can accurately obtain nucleic acid detection results in the amplification container.

[0017] According to an embodiment of the present invention, the method for classifying isothermal amplification containers may further include at least one of the following technical features:

[0018] According to an embodiment of the present invention, for a given amplification container, the first classification is performed by: determining the first signal threshold of the amplification based on the signal at at least one initial time point; and classifying the amplification container between the amplification container and the non-amplification container based on the first difference between the signal at other time points of the amplification container and the first signal threshold. For a given amplification container, the second classification is performed by: determining the second signal threshold based on the corrected signal at at least one initial time point; and classifying the amplification container between the amplification container and the non-amplification container based on the second difference between the signal at other time points of the amplification container and the second signal threshold. The method of two classifications can analyze signal data at different levels, improve the accuracy and stability of classification, reduce the error rate, and make the final classification result more reliable and accurate.

[0019] In some examples of the present application, the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0020] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool. In the present application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0021] According to an embodiment of the present invention, the amplification baseline can be obtained by calculation methods such as fitting, interpolation, regression or averaging.

[0022] According to an embodiment of the present invention, the first signal threshold S_thresh is obtained by the following calculation formula:

[0023] S_thresh=AVG+N1×STD

[0024] Among them, AVG represents the average value of the original real-time signal data selected for a classification; STD represents the standard deviation of the original real-time signal data selected for a classification; N1 is an integer.

[0025] In some examples of the present application, in the aforementioned first signal threshold calculation formula, N1 includes integers of 1 to 25. Specifically, N1 includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N1 is 10.

[0026] It should be noted that the above signal threshold calculation method is only exemplary. In addition, the signal threshold can also be calculated by variance or coefficient of variation.

[0027] According to an embodiment of the present invention, the first difference is determined by comparing the signal of the amplification container at other time points with the first signal threshold.

[0028] According to an embodiment of the present invention, the signal at other time points is greater than the first signal threshold, which is an indication that the amplification container is a preliminarily judged amplification container; the signal at at least one other time point is less than the first signal threshold, which is an indication that the amplification container is a preliminarily judged non-amplification container.

[0029] It should be noted that the initial judgment of amplification (initial judgment of positive amplification) in the present application is obtained through comprehensive judgment, that is, as long as the signal result at one time point is greater than the first signal threshold, it is considered to be initial judgment of positive amplification; similarly, the initial judgment of non-amplification (initial judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results at all time points are less than the first signal threshold.

[0030] According to an embodiment of the present invention, the second signal threshold S norm _thresh is obtained by the following calculation formula:

[0031] S norm _thresh=AVG norm +N2×STD norm

[0032] Among them, AVG norm Indicates the average value of the corrected real-time signal data selected for secondary classification; STD norm Represents the standard deviation of the corrected real-time signal data selected for secondary classification; N2 is an integer.

[0033] In some examples of the present application, in the aforementioned second signal threshold calculation formula, N2 takes values ​​including integers of 1 to 25. Specifically, N2 takes values ​​including: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N2 takes a value of 10.

[0034] It should be noted that the above signal threshold calculation method is only exemplary. In addition, the signal threshold can also be calculated by variance or coefficient of variation.

[0035] In some examples of the present application, the values ​​of N1 and N2 may be the same or different, and are generally set based on actual experimental requirements.

[0036] According to an embodiment of the present invention, the second difference is determined by comparing the signal of the amplification container at other time points with the second signal threshold.

[0037] According to an embodiment of the present invention, the signal at other time points is greater than the second signal threshold, which is an indication that the amplification container is a final-judgment amplification container; the signal at other time points is less than the second signal threshold, which is an indication that the amplification container is a final-judgment non-amplification container.

[0038] It should be noted that the final judgment of amplification (final judgment of positive amplification) described in the present application is obtained through comprehensive judgment, that is, the signal result of any one of the other time points is greater than the second signal threshold, which is considered to be the final judgment of positive amplification; similarly, the final judgment of non-amplification (final judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results of other time points are all less than the second signal threshold.

[0039] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0040] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer amplification and helicase-dependent amplification.

[0041] In the second aspect of the present invention, the present invention proposes a method for determining the positive time (TTP value) of an amplification reaction. According to an embodiment of the present invention, the method includes: performing an amplification reaction in a plurality of amplification containers; classifying the plurality of amplification containers according to the method of the first aspect of the present invention, determining the final judgment amplification container and the final judgment non-amplification container; constructing a data function of the corrected real-time signal relative to the sampling time based on at least a portion of the plurality of amplification containers; determining the signal threshold of the final judgment non-amplification container based on the corrected real-time signal of the final judgment non-amplification container; and determining the positive time of a given amplification container corresponding to the signal threshold based on the data function. According to an embodiment of the present invention, the method improves the determination accuracy and experimental efficiency of the positive time (TTP value) of the isothermal amplification reaction through automated and accurate data processing, while also reducing resource waste.

[0042] In some examples of the present application, the TTP value can also be used to measure parameters such as sensitivity, specificity, precision, repeatability, reproducibility, and detection limit, so that independent test results across amplification systems, instrument platforms, and laboratories have intra-batch and inter-batch result data comparability.

[0043] According to an embodiment of the present invention, the method for determining the positive time of an amplification reaction may further include at least one of the following technical features:

[0044] According to an embodiment of the present invention, the data function can be calculated and obtained by polynomial fitting, spline fitting, exponential fitting or other equivalent methods.

[0045] In some examples of the present application, the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0046] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool. In the present application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0047] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0048] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer amplification and helicase-dependent amplification.

[0049] In the third aspect of the present invention, the present invention proposes a method for analyzing biological samples by amplification reaction. According to an embodiment of the present invention, the method includes: obtaining a biological sample suspected of containing nucleic acid; performing an amplification reaction in multiple amplification containers for the biological sample; determining the positive reporting time of a given amplification container according to the method of the second aspect of the present invention; and analyzing the nucleic acid content in the biological sample based on the positive reporting time. According to an embodiment of the present invention, the method has the advantages of high sensitivity, high efficiency and rapidity, low cost, easy automation and wide adaptability, making it a very valuable analytical tool in the fields of biological research, medical diagnosis and environmental monitoring. In some examples of the present application, the method can also be applied to the analysis of different biological samples (such as the expression level of the target gene, the efficiency of the kit or primer, the sample purity and dilution degree or the comparison between samples, etc.), and has the advantages of short analysis time and high sensitivity.

[0050] According to an embodiment of the present invention, the above method for analyzing a biological sample by an amplification reaction may further include at least one of the following technical features:

[0051] In some examples of the present application, the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0052] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool. In the present application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0053] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0054] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer amplification and helicase-dependent amplification.

[0055] In a fourth aspect of the present invention, the present invention proposes a method for determining the nucleic acid content in a nucleic acid sample. According to an embodiment of the present invention, the method comprises: performing an amplification reaction in a plurality of amplification containers for the nucleic acid sample; determining the positive reporting time of a given amplification container according to the method of the second aspect of the present invention; and determining the content of nucleic acid in the nucleic acid sample based on the positive reporting time. According to an embodiment of the present invention, the method has the advantages of high accuracy, good real-time performance, wide adaptability, simple operation and high-throughput processing, making it a reliable and efficient method for determining the nucleic acid content in a nucleic acid sample.

[0056] In some examples of the present application, the method can determine the content of nucleic acid in the test nucleic acid sample based on the positive reporting time, and the method has a wide applicability and can be used to parallel compare the test results obtained based on different labeling methods; it is also suitable for analyzing the limitations of result judgment caused by differences in detection procedures in the same labeling method.

[0057] According to an embodiment of the present invention, the method for determining the nucleic acid content in a nucleic acid sample may further include at least one of the following technical features:

[0058] In some examples of the present application, the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0059] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool. In the present application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0060] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0061] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer amplification and helicase-dependent amplification.

[0062] In a fifth aspect of the present invention, the present invention provides a method for determining the content of a target nucleic acid molecule in a nucleic acid sample. According to an embodiment of the present invention, the method comprises: using primers for the target nucleic acid molecule, performing an isothermal amplification reaction in multiple isothermal amplification containers; determining the positive reporting time of a given amplification container according to the method described in the second aspect; and determining the content of the target nucleic acid molecule in the nucleic acid sample based on the positive reporting time.

[0063] According to an embodiment of the present invention, the method can quickly calculate the expression level of the target gene in the test nucleic acid sample based on the positive reporting time.

[0064] According to an embodiment of the present invention, the method for determining the content of target nucleic acid molecules in a nucleic acid sample may further include at least one of the following technical features:

[0065] According to an embodiment of the present invention, the amplification container is selected from at least one of a single PCR tube, an 8-well tube, a 96-well or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0066] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool. In the present application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0067] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.

[0068] In the sixth aspect of the present invention, the present invention proposes a device for classifying isothermal amplification containers. According to an embodiment of the present invention, the model structure includes: a data collection module, which is used to collect signals at multiple given time points for each of the multiple amplification containers, so as to obtain an original real-time signal data set; a first classification determination module, the first classification predetermined module is connected to the data collection module, and is used to perform a classification for at least one of the multiple amplification containers based on the original real-time signal data set, so as to obtain a preliminary judgment amplification container and a preliminary judgment non-amplification container; a correction module, the correction module is connected to the first classification determination module, and is used to determine the amplification baseline based on at least a part of the signal of the preliminary judgment non-amplification container, and use the amplification baseline to correct at least a part of the original real-time signal data set, so as to obtain a corrected real-time signal data set; a second classification determination module, the second classification determination module is connected to the correction module, and is used to perform a secondary classification for at least one of the multiple amplification containers based on the corrected real-time signal data set, so as to obtain a final judgment amplification container and a final judgment non-amplification container. According to an embodiment of the present invention, the device can automatically collect the time point signal data of the sample to be tested in the isothermal amplification container without manual intervention, which can improve the experimental efficiency. In addition, the double classification determines positive amplification and negative amplification, which improves the accuracy of classification. Based on machine learning technology, the above-mentioned equipment can also perform self-learning and optimization to adapt to isothermal amplification container classification tasks under different conditions.

[0069] It should be noted that if Figure 1 As shown, the data collection module S100 is connected to the first classification determination module S200, the first classification determination module S200 is connected to the correction module S300, and the correction module S300 is connected to the second classification determination module S400.

[0070] According to an embodiment of the present invention, the device for classifying amplification containers may further include at least one of the following technical features:

[0071] According to an embodiment of the present invention, for a given amplification container, the first classification is performed by: determining the first signal threshold of the amplification based on the signal at at least one initial time point; and classifying the amplification container between the amplification container and the non-amplification container based on the first difference between the signal at other time points of the amplification container and the first signal threshold. For a given amplification container, the second classification is performed by: determining the second signal threshold based on the corrected signal at at least one initial time point; and classifying the amplification container between the amplification container and the non-amplification container based on the second difference between the signal at other time points of the amplification container and the second signal threshold. The method of two classifications can analyze signal data at different levels, improve the accuracy and stability of classification, reduce the error rate, and make the final classification result more reliable and accurate.

[0072] In some examples of the present application, the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0073] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool. In the present application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0074] According to an embodiment of the present invention, the amplification baseline can be obtained by calculation methods such as fitting, interpolation, regression or averaging.

[0075] According to an embodiment of the present invention, the first signal threshold S_thresh is obtained by the following calculation formula:

[0076] S_thresh=AVG+N1×STD

[0077] Among them, AVG represents the average value of the original real-time signal data selected for a classification; STD represents the standard deviation of the original real-time signal data selected for a classification; N1 is an integer.

[0078] In some examples of the present application, in the aforementioned first signal threshold calculation formula, N1 includes integers of 1 to 25. Specifically, N1 includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N1 is 10.

[0079] It should be noted that the above signal threshold calculation method is only exemplary. In addition, the signal threshold can also be calculated by variance or coefficient of variation.

[0080] According to an embodiment of the present invention, the first difference is determined by comparing the signal of the amplification container at other time points with the first signal threshold.

[0081] According to an embodiment of the present invention, the signal at other time points is greater than the first signal threshold, which is an indication that the amplification container is a preliminarily judged amplification container; the signal at at least one other time point is less than the first signal threshold, which is an indication that the amplification container is a preliminarily judged non-amplification container.

[0082] It should be noted that the initial judgment of amplification (initial judgment of positive amplification) in the present application is obtained through comprehensive judgment, that is, as long as the signal result at one time point is greater than the first signal threshold, it is considered to be initial judgment of positive amplification; similarly, the initial judgment of non-amplification (initial judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results at all time points are less than the first signal threshold.

[0083] According to an embodiment of the present invention, the second signal threshold S norm _thresh is obtained by the following calculation formula:

[0084] S norm _thresh=AVG norm +N2×STD norm

[0085] Among them, AVG norm Indicates the average value of the corrected real-time signal data selected for secondary classification; STD norm Represents the standard deviation of the corrected real-time signal data selected for secondary classification; N is an integer.

[0086] In some examples of the present application, in the aforementioned second signal threshold calculation formula, N2 takes values ​​including integers of 1 to 25. Specifically, N2 takes values ​​including: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N2 takes a value of 10.

[0087] It should be noted that the above signal threshold calculation method is only exemplary. In addition, the signal threshold can also be calculated by variance or coefficient of variation.

[0088] According to an embodiment of the present invention, the second difference is determined by comparing the signal of the amplification container at other time points with the second signal threshold.

[0089] According to an embodiment of the present invention, the signal at other time points is greater than the second signal threshold, which is an indication that the amplification container is a final-judgment amplification container; the signal at other time points is less than the second signal threshold, which is an indication that the amplification container is a final-judgment non-amplification container.

[0090] It should be noted that the final judgment of amplification (final judgment of positive amplification) described in the present application is obtained through comprehensive judgment, that is, the signal result of any one of the other time points is greater than the second signal threshold, which is considered to be the final judgment of positive amplification; similarly, the final judgment of non-amplification (final judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results of other time points are all less than the second signal threshold.

[0091] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0092] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer amplification and helicase-dependent amplification.

[0093] In the seventh aspect of the present invention, the present invention proposes a system for determining the time of reporting positive isothermal amplification reaction. According to an embodiment of the present invention, the system includes: an amplification reaction device, the amplification reaction device is used to perform an amplification reaction; and the device described in the sixth aspect, the device is connected to the multiple amplification containers, and is used to classify the amplification containers; and a data function construction device, the data function construction device is connected to the device described in the sixth aspect, and is used to construct a data function of the corrected real-time signal relative to the sampling time based on at least a part of the multiple amplification containers; and a signal threshold determination device for a final non-amplification container, the signal threshold determination device for the final non-amplification container is connected to the data function construction device, and is used to determine the signal threshold of the final non-amplification container based on the corrected real-time signal of the final non-amplification container; and a reporting positive time acquisition device, the reporting positive time acquisition device is connected to the signal threshold determination device and the data function construction device of the final non-amplification container, and is used to determine the reporting positive time of a given amplification container corresponding to the signal threshold based on the data function. According to an embodiment of the present invention, the system for determining the positive reporting time of the isothermal amplification reaction can accurately judge positive samples and negative samples, and can evaluate parameters such as sensitivity, specificity, precision, repeatability, reproducibility, and detection limit based on the obtained positive reporting time data, so that independent test results across amplification systems, instrument platforms, and laboratories have intra-batch and inter-batch result data comparability.

[0094] It should be noted that if Figure 2As shown, the amplification reaction device S500 is connected to the device A01 for classifying amplification containers, the device A01 for classifying amplification containers is connected to the data function construction device S700, the data function construction device S700 is connected to the signal threshold determination device S800 for final judgment of non-amplification containers, and the signal threshold determination device S800 for final judgment of non-amplification containers is connected to the positive reporting time acquisition device S900.

[0095] In the eighth aspect of the present invention, the present invention proposes a system for analyzing biological samples by isothermal amplification reaction. According to an embodiment of the present invention, the system includes: a biological sample acquisition device, the biological sample acquisition device is used to acquire a biological sample suspected of containing nucleic acid; and the system for determining the positive reporting time of an amplification reaction as described in the seventh aspect of the present invention, the system is connected to the biological sample acquisition device, and is used to determine the positive reporting time of a given amplification container; and an analysis device, the analysis device is connected to the system for determining the positive reporting time of an amplification reaction, and is used to analyze the nucleic acid content in the biological sample.

[0096] According to an embodiment of the present invention, the advantage of the system for analyzing biological samples by isothermal amplification reaction is that it avoids the complicated data analysis process in traditional PCR technology, and the model structure can analyze biological samples quickly, accurately and automatically.

[0097] It should be noted that if Figure 3 As shown, the biological sample acquisition device S001 is connected to the system X01 for determining the positive reporting time of the amplification reaction (including an amplification reaction device S500, a device S600 for classifying amplification containers, a data function construction device S700, a signal threshold determination device S800 for final judgment of non-amplification containers, and a positive reporting time acquisition device S900), and the system X01 for determining the positive reporting time of the amplification reaction is connected to the analysis device S002.

[0098] In the ninth aspect of the present invention, the present invention provides a system for determining the nucleic acid content in a nucleic acid sample. According to an embodiment of the present invention, the system comprises: the system for determining the positive reporting time of an amplification reaction as described in the seventh aspect of the present invention, which is used to determine the positive reporting time of a given amplification container; and a nucleic acid content acquisition device, which is connected to the system and is used to determine the nucleic acid content in the nucleic acid sample.

[0099] According to an embodiment of the present invention, the system can determine the content of nucleic acid in a nucleic acid sample by the positive reporting time of an isothermal amplification reaction, thereby avoiding tedious data analysis processes and saving time and costs.

[0100] It should be noted that if Figure 4As shown, the system X01 for determining the positive reporting time of an amplification reaction (including an amplification reaction device S500, a device S600 for classifying amplification containers, a data function construction device S700, a signal threshold determination device S800 for final judgment of non-amplification containers, and a positive reporting time acquisition device S900) is connected to the nucleic acid content acquisition device S003.

[0101] In the tenth aspect of the present invention, the present invention proposes a system for determining the content of target nucleic acid molecules in a nucleic acid sample. According to an embodiment of the present invention, the system comprises: the system for determining the positive reporting time of isothermal amplification reaction as described in the seventh aspect of the present invention, which is used to determine the positive reporting time of a given isothermal amplification container; and a target nucleic acid molecular weight acquisition device, which is connected to the system and is used to determine the content of the target nucleic acid molecule in the nucleic acid sample.

[0102] According to an embodiment of the present invention, the system can automatically determine the content of target nucleic acid molecules in a nucleic acid sample.

[0103] It should be noted that if Figure 5 As shown, the system X01 for determining the positive reporting time of an amplification reaction (including an amplification reaction device S500, a device S600 for classifying amplification containers, a data function construction device S700, a signal threshold determination device S800 for final judgment of non-amplification containers, and a positive reporting time acquisition device S900) is connected to the target nucleic acid molecular weight acquisition device S004.

[0104] In an eleventh aspect of the present invention, the present invention provides a computer program product. According to an embodiment of the present invention, the computer program product includes computer instructions, and when part or all of the computer instructions are run on a computer, the method described in the first aspect, the second aspect, the third aspect, the fourth aspect, or the fifth aspect of the present invention is executed.

[0105] It should be noted that the computer program product includes analysis application software or a program compression package.

[0106] In a twelfth aspect of the present invention, the present invention provides a computing device. According to an embodiment of the present invention, the computing device comprises: a memory and a processor; the memory is used to store a computer program; the processor is used to execute the computer program to implement the method described in the first aspect, the second aspect, the third aspect, the fourth aspect, or the fifth aspect of the present invention.

[0107] In a thirteenth aspect of the present invention, the present invention provides a computer-readable storage medium. According to an embodiment of the present invention, the storage medium includes computer instructions, and when the instructions are executed by a computer, the computer implements the method described in the first aspect, the second aspect, the third aspect, the fourth aspect, or the fifth aspect of the present invention.

[0108] It should be noted that various implementations of the systems and techniques described above in this article can be implemented in digital electronic circuit systems, integrated circuit systems, FPGAs (Field Programmable Gate Arrays), ASICs (Application-Specific Integrated Circuits), ASSPs (Application-Specific Standard Products), SOCs (System On Chips), CPLDs (Complex Programmable Logic Devices), computer hardware, firmware, software, and / or combinations thereof. These various implementations may include: being implemented in one or more computer programs, which may be executed and / or interpreted on a programmable system including at least one programmable processor, which may be a dedicated or general-purpose programmable processor, which may receive data and instructions from a storage system, at least one input device, and at least one output device, and transmit data and instructions to the storage system, the at least one input device, and the at least one output device.

[0109] The program code for implementing the method disclosed in the present application can be written in any combination of one or more programming languages. These program codes can be provided to a processor or controller of a general-purpose computer, a special-purpose computer, or other programmable data processing device, so that the program code, when executed by the processor or controller, enables the functions / operations specified in the flow chart and / or block diagram to be implemented. The program code can be executed entirely on the machine, partially on the machine, partially on the machine as a stand-alone software package and partially on a remote machine, or entirely on a remote machine or server.

[0110] In the context disclosed in the present application, a machine-readable medium may be a tangible medium that may contain or store a program for use by or in conjunction with an instruction execution system, device, or equipment. A machine-readable medium may be a machine-readable signal medium or a machine-readable storage medium. A machine-readable medium may include, but is not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, device, or device, or any suitable combination of the foregoing. More specific examples of machine-readable storage media may include electrical connections based on one or more lines, portable computer disks, hard disks, RAM, ROM, EPROM (Electrically Programmable Read-Only-Memory) or flash memory, optical fiber, CD-ROM (Compact Disc Read-Only Memory), optical storage devices, magnetic storage devices, or any suitable combination of the foregoing.

[0111] To provide interaction with a user, the systems and techniques described herein can be implemented on a computer having: a display device (e.g., a CRT (Cathode-Ray Tube) or LCD (Liquid Crystal Display) monitor) for displaying information to the user; and a keyboard and pointing device (e.g., a mouse or trackball) through which the user can provide input to the computer. Other types of devices can also be used to provide interaction with the user; for example, the feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user can be received in any form (including acoustic input, voice input, or tactile input).

[0112] The systems and techniques described herein may be implemented in a computing system that includes backend components (e.g., as a data server), or a computing system that includes middleware components (e.g., an application server), or a computing system that includes frontend components (e.g., a user computer with a graphical user interface or a web browser through which a user can interact with implementations of the systems and techniques described herein), or a computing system that includes any combination of such backend components, middleware components, or frontend components. The components of the system may be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include: LAN (Local Area Network), WAN (Wide Area Network), the Internet, and blockchain networks.

[0113] A computer system may include a client and a server. The client and the server are generally remote from each other and usually interact through a communication network. The relationship between the client and the server is generated by computer programs running on the corresponding computers and having a client-server relationship with each other. The server may be a cloud server, also known as a cloud computing server or cloud host, which is a host product in the cloud computing service system to solve the defects of difficult management and weak business scalability in traditional physical hosts and VPS services ("Virtual Private Server", or "VPS" for short). The server may also be a server of a distributed system, or a server combined with a blockchain.

[0114] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solutions disclosed in this disclosure can be achieved, and this document does not limit this.

[0115] It should be noted that the features and technical effects described in this article for different aspects can be used as reference for each other and will not be repeated here.

[0116] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0118] Figure 1 is a diagram of a device for classifying amplification containers according to an embodiment of the present invention;

[0119] Figure 2 2 is a schematic diagram of a system for determining the positive time of an amplification reaction according to an embodiment of the present invention;

[0120] Figure 3 is a schematic diagram of a system for analyzing biological samples through an amplification reaction according to an embodiment of the present invention;

[0121] Figure 4 is a schematic diagram of a system for determining the nucleic acid content in a nucleic acid sample according to an embodiment of the present invention;

[0122] Figure 5 is a schematic diagram of a system for determining the content of a target nucleic acid molecule in a nucleic acid sample according to an embodiment of the present invention;

[0123] Figure 6The real-time signal curve processing result according to Example 1 of the present invention; wherein, (A) the original signal curve; (B) step c, the positive original signal curve; (C) step d, the negative original signal curve; (D) step e, the polynomial fitting average baseline graph; (E) step e, the correction and initialization signal curve graph. (F) steps e and h, the normalized signal curve spline fitting graph.

[0124] Figure 7 is the real-time signal curve processing result according to Example 1 of the present invention; wherein, (A) the real-time signal curve processing result of well position A01; (B) the real-time signal curve processing result of well position A08; (C) the real-time signal curve processing result of well position A12; (D) the TTP value data table of a 96-well PCR plate;

[0125] Figure 8 is a summary diagram of the real-time signal curve processing results of each well position of the 96-well plate according to Example 1 of the present invention;

[0126] Fig. 9 is the dual-channel LAMP real-time signal curve according to Example 2 of the present invention (Bori FQD-96A original data);

[0127] Fig.10 is the result of the dual-channel real-time signal curve processing according to Example 2 of the present invention; wherein, (A) the original signal curve of the FAM channel; (B) the signal curve after the FAM channel is processed; (C) the original signal curve of the ROX channel; (D) the signal curve after the ROX channel is processed;

[0128] Fig.11 It is the fluorescence signal image of the endpoint method of the 96-well PCR plate according to Example 2 of the present invention; wherein, (A) is a dark field fluorescence image; and (B) is a bright field fluorescence image. DETAILED DESCRIPTION

[0129] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0130] Definition and Description

[0131] As used herein, unless otherwise indicated, the singular forms "a," "an," and the like include plural referents (more than one); "a set" or "a plurality" refers to two or more.

[0132] In this document, unless otherwise specified, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0133] In this document, unless otherwise specified, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated; features specified as "first", "second", etc. may explicitly or implicitly include one or more of the said features.

[0134] In this article, the real-time signal fitting method corresponding to the negative amplifier includes but is not limited to polynomial fitting (the polynomial order is set as required), and other equivalent methods that can achieve the same effect are applicable.

[0135] In this article, the term "loop-mediated isothermal amplification (LAMP)" is a nucleic acid amplification method that can specifically and rapidly amplify DNA molecules under constant temperature (60-68°C). Compared with traditional PCR technology, LAMP technology has the advantages of high specificity, high sensitivity, simple operation, and no need for expensive instruments and equipment. Its principle is to use a set of specific primers to amplify the target DNA sequence under constant temperature conditions, and detect the amplified product by forming a fluorescent signal or observing the color change with the naked eye.

[0136] At present, the sequence-specific labeling methods used in loop-mediated amplification (LAMP) technology mainly include fluorescence of loop primer upon self-dequenching, HyBeacon probe, guanine quenching probe, graphene oxide based fluorescence resonance energy transfer (GO-FRET), detection of amplification by release of quenching (DARQ), quenching of unincorporated amplification signal reporters (QUASR), one-step strand displacement (OSD), molecular beacon, light cycler probe, assimilating probe, mediator displacement probe, etc. Different labeling methods are divided into two technical schemes: endpoint detection and real-time detection. Since the principles, technical solutions, operating procedures and detection equipment and instruments on which these methods are based are different, there is a lack of common technical standards and they cannot meet the requirements of standardization, automation and high throughput in nucleic acid detection scenarios. Therefore, the present invention proposes a method for classifying amplification containers. To determine whether the designated amplification container is positive amplification or negative amplification, on this basis, the reporting time (TTP value) of positive amplification is determined in combination with the time point information.

[0137] In one example of the present application, an isothermal amplification container is used to perform an isothermal amplification reaction to determine whether the container is positively amplified. If it is positively amplified, a method for determining the positive reporting time is provided.

[0138] Specifically, for each of the multiple isothermal amplification containers, signal collection is performed at multiple given time points to obtain an original real-time signal data set; based on the original real-time signal data set, at least one of the multiple isothermal amplification containers is classified once to obtain a preliminary amplification container and a preliminary non-amplification container; based on at least a portion of the signal of the preliminary non-amplification container, an isothermal amplification baseline is determined, and the isothermal amplification baseline is used to correct at least a portion of the original real-time signal data set to obtain a corrected real-time signal data set; based on the corrected real-time signal data set, a secondary classification is performed on at least one of the multiple isothermal amplification containers to obtain a final amplification container and a final non-amplification container.

[0139] It should be noted that the determination of the amplification baseline comes from all negative data in the same channel. For example, in an 8 (rows) × 12 (columns) PCR plate, the first row is used for amplification test of the FAM (fluorescent probe) channel, and the second row is used for amplification test of the ROX (fluorescent probe) channel. The amplification baseline of the FAM channel is calculated from the negative data in the first row, and the amplification baseline of the ROX channel is calculated from the negative data in the second row. If all data in the PCR plate come from the same channel, then the amplification baseline of the channel is calculated from all negative data in the PCR plate.

[0140] Wherein, for a given isothermal amplification container, the primary classification is performed by:

[0141] Based on the signal of at least one initial time point, a first signal threshold of the isothermal amplification is determined; and based on the difference between the signal of other time points of the isothermal amplification container and the first signal threshold, the isothermal amplification container is classified between the amplification container and the non-amplification container. For a given isothermal amplification container, the secondary classification is performed by the following steps: based on the corrected signal of at least one initial time point, a second signal threshold is determined; and based on the difference between the signal of other time points of the isothermal amplification container and the second signal threshold, the isothermal amplification container is secondary classified between the amplification container and the non-amplification container to determine the final judgment amplification container (final judgment positive amplification) and the final judgment non-amplification container (final judgment negative amplification);

[0142] Based on at least a portion of the multiple isothermal amplification containers, a data function of the corrected real-time signal relative to the sampling time is constructed; based on the corrected real-time signal of the final judgment non-amplification container, a signal threshold of the final judgment non-amplification container is determined; and based on the data function, a reporting time (TTP value) of a given amplification container corresponding to the signal threshold is determined.

[0143] Specifically, for ease of understanding, the technical solution of the present application (taking self-circulating chain displacement amplification (LAMP) real-time signal data analysis as an example) is explained and illustrated in detail below. The specific steps of the technical solution are as follows:

[0144] 1) Raw data acquisition and storage: Prepare a series of amplification detection systems according to different types of samples to be tested and pack them in specific amplification containers, start self-circulating chain displacement amplification (LAMP) on a machine (real-time fluorescence thermal cycle or real-time fluorescence constant temperature equipment, such as Hangzhou Biore FQD-96A, Shanghai Hongshi SLAN-96S, American Thermo Fisher ABI-7500, etc.), obtain the detection equipment according to the preset real-time detection temperature control and signal acquisition program, obtain the raw real-time signal data S (S contains A×T real-time signal data) of all amplifiers (amplifier refers to the smallest amplification unit in the amplification container, such as each single PCR tube, or a single tube in an 8-well row of tubes, a single well in a 96- or 384-well PCR plate, etc., the total number of amplifiers is recorded as A) of each channel of the real-time detection instrument and equipment at a total of T signal acquisition time points, and save the data matrix in the storage medium;

[0145] In some examples of the present application, after the original data is collected and stored, data expansion can be performed selectively. The so-called data expansion includes: using a computer device to read the data matrix in the storage medium to obtain the original signal data of all amplifiers in a specific amplification container. According to the T real-time signal data (A1, A2, ..., AT) corresponding to each amplifier, calculate (using fitting, interpolation, regression or averaging calculation methods) to obtain each continuous real-time signal curve data or expand it to (T+E) real-time signal data (B1, B2, ..., BT+E) with higher data density, where E is a positive integer. To distinguish it from the original real-time signal data S, the expanded real-time signal data can be recorded as S'.

[0146] 2) Primary classification judgment: The computer device reads the data matrix in the storage medium and obtains the original signal data of all amplifiers in the specific amplification container. According to the initial N data (A1, A2, ..., A N , N is a preset value, such as N can be 5 or 6, etc.), calculate the signal threshold S_thresh of each amplifier or all amplifiers, and perform the following N+1, N+2, ..., T, a total of (TN) real-time signal data (A N+1 , A N+2 , …, A T ) is compared with the signal threshold S_thresh to determine whether the amplifier has amplified (if the threshold is larger, it is determined that no amplification has occurred, and if the threshold is smaller, it is determined that amplification has occurred). The above steps are performed on all amplifiers in sequence and the determination is completed. If no amplification occurs, it is determined as a negative result, and if amplification occurs, it is determined as a positive result;

[0147] 3) Calibration and initial normalization: According to the experimental group of negative amplifier (A neg Group) real-time signal data, get A neg ×T real-time signal data, and calculate (using fitting, interpolation, regression or averaging methods) the average baseline S of the unamplified real-time signal data base , determine the tilt of the signal background. According to the original real-time signal data S of all amplifiers and the average baseline S without amplification base , calculate the baseline corrected real-time signal data S flat Then, the baseline-corrected real-time signal data of all amplifiers is initialized (the baseline-corrected real-time signal data S of each amplifier is initialized). flat According to the alignment of the starting data points to make the data comparable) and (linear) normalization processing, all signal data values ​​are normalized to [0,1] to obtain the initial normalized real-time signal data S norm ;

[0148] 4) Secondary classification and positive time (TTP value) calculation: Use the method in step 2) to calculate the initial normalized real-time signal data S of each amplifier or all amplifiers. norm The signal threshold S norm _thresh, all initial normalized real-time signal data of each amplifier and the signal threshold S norm _thresh, and then the amplification results of all amplifiers are judged again. If no amplification is judged as a negative result, and if amplification is judged as a positive result, a suitable model (such as spline fitting, piecewise linear interpolation, local linear regression, kernel regression or other equivalent methods) is selected to calculate the initial normalized real-time signal S of all amplifiers. norm Fitting and interpolation are performed to obtain the data function Fit(S) of each amplifier real-time signal about T signal acquisition time points. norm ). Then, determine the data function Fit(S of each positive amplifier norm ) and signal threshold S norm The unique intersection of _thresh is calculated, and the signal acquisition time value corresponding to the intersection is calculated to obtain the positive reporting time (TTP value) of the amplifier. The data function Fit (S norm ) and signal threshold S norm _thresh has no intersection point, so the positive reporting time (TTP value) is set to infinity (Inf).

[0149] It should be noted that the above analysis method is not limited to different labeling methods, operation procedures and detection equipment and instruments. Through calibration and initial normalization, the above method can automatically process and determine the real-time signal data of each amplifier with different sample types but the same or similar biochemical system, and calculate the universal quantifiable indicator positive reporting time to characterize the appearance of positive signals, thereby realizing semi-quantitative or quantitative analysis of the detection results.

[0150] Embodiments of the present invention will be described in more detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but are not to be construed as limitations of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not indicate manufacturers are conventional products that can be obtained commercially.

[0151] Example 1: Algorithm verification based on single-channel loop-mediated isothermal amplification real-time signal

[0152] 1) LAMP real-time signal data collection

[0153] According to an embodiment of the present invention, in order to verify the feasibility of the real-time signal result determination method described in the technical solution of the present invention, this embodiment adopts a loop-mediated isothermal amplification (LAMP) experimental method, and a single-channel detection of nucleic acid templates containing several different concentrations of MGI (MGI) self-developed double-stranded probe system is prepared. The total volume of the detection system is 30μl, which contains 12μl of template. The amplification container is a 96-well PCR plate, which contains 6 positive quality controls of 2 different conditions (condition 1: sample diluted with nuclease-free water, condition 2: sample diluted with sample preservation solution), 10 negative quality controls of 2 different conditions (condition 1: sample diluted with nuclease-free water, condition 2: sample diluted with sample preservation solution), 80 simulated samples of 4 low-concentration nucleic acid templates, and the concentrations of the preservation solution samples before extraction are 500, 300, 200, and 100 copies / ml, respectively, and each concentration contains 20 technical replicates. The real-time signal acquisition device is Hangzhou Biori FQD-96A, running the device client software Gene-9660, and setting the acquisition program to 35 time points in three stages: pre-acquisition, reverse transcription, and isothermal amplification. The temperature is set to 30°C during signal acquisition, and the temperature is set to 65°C during reverse transcription and isothermal amplification. Click "Start Run" to start the acquisition program. Until the acquisition program completes 35 acquisition actions and obtains all amplified real-time signal data, the client software Gene-9660 will automatically draw the acquired data synchronously. Figure 6Since the client software Gene-9660 does not have the function of analyzing LAMP real-time signals, the software can be used to export the raw data of LAMP real-time signals to Excel or text format files and save them for data analysis.

[0154] 2) LAMP real-time signal data analysis

[0155] Based on the technical solution disclosed in the present invention, the LAMP real-time signal raw data is processed and analyzed. The specific steps are as follows:

[0156] a. Read the LAMP real-time signal raw data from the Excel or text format file in the storage medium, and generate a two-dimensional matrix of the real-time signal raw data according to the two dimensions of signal acquisition time point and amplified real-time signal value (the matrix contains 96×35 real-time signal data);

[0157] b. Take the real-time signal values ​​of the first six signal acquisition time points to calculate the signal threshold of each amplifier (a total of 96). The calculation steps are as follows:

[0158] First, the average value (AVG) and standard deviation (STD) of the real-time signal values ​​of the first six signal acquisition time points of each amplifier were calculated;

[0159] Secondly, the signal threshold of each amplifier is calculated based on the formula (S_thresh = AVG + N1 × STD, N1 is 10);

[0160] c. Obtain the maximum value of the last 29 real-time signal values ​​of each amplifier (35 minus the first 6 signal values), compare the value with the corresponding signal threshold S_thresh, and record the threshold comparison result (record 1 if greater than the threshold, and record 0 if less than the threshold). Based on the threshold comparison result, the amplifier with a value of 1 is preliminarily determined as positive (amplified), and the amplifier with a value of 0 is preliminarily determined as negative (not amplified);

[0161] d. The corresponding real-time signal values ​​of all negative amplifiers are fitted with a polynomial (the polynomial order is set to 8) to calculate the average baseline of the unamplified real-time signal data. Based on the above average baseline data, the two-dimensional matrix of the real-time signal raw data is processed to obtain the two-dimensional matrix of the real-time signal data after baseline correction (the matrix contains 96×35 real-time signal data);

[0162] e. Initialize the two-dimensional matrix of real-time signal data after baseline correction according to the real-time signal value at the first signal acquisition time point (data alignment), and normalize all signal data values ​​of the two-dimensional matrix to [0, 1] to obtain an initial normalized two-dimensional matrix of real-time signal data;

[0163] f. According to the two-dimensional matrix of initial normalized real-time signal data, take the initial normalized real-time signal values ​​of the first 6 signal acquisition time points to calculate the initial normalized signal threshold of each amplifier (a total of 96). The calculation steps are as follows:

[0164] First, the average of the initial normalized real-time signal values ​​(AVG) of the first six signal acquisition time points of each amplifier was calculated. norm ) and standard deviation (STD norm );

[0165] Secondly, based on the formula (S norm _thresh=AVG norm +N2×STD norm , N2 is 10) Calculate the initial normalized signal threshold S of each amplifier norm _thresh;

[0166] g. Calculate the maximum value of the last 29 initial normalized real-time signal values ​​of each amplifier (35 minus the first 6 signal values), and compare it with the corresponding initial normalized signal threshold S norm _thresh compares the size and records the threshold comparison result (greater than the threshold is recorded as 1, less than the threshold is recorded as 0). Based on the threshold comparison result, the amplifier with a value of 1 is preliminarily judged as positive (amplified), and the amplifier with a value of 0 is preliminarily judged as negative (not amplified);

[0167] h. Use the smoothing spline fitting method to fit the initial normalized real-time signal data of all amplifiers to obtain the real-time signal smooth curve corresponding to each amplifier, determine the unique intersection of the curve and the corresponding initial normalized signal threshold, and calculate the signal acquisition time value corresponding to the intersection, which is the positive reporting time (TTP value) of the amplifier. Since there is no intersection between the real-time signal smooth curve of each negative amplifier and the corresponding threshold, its positive reporting time (TTP value) is set to infinity (Inf);

[0168] i. Finally, the real-time signal curve processing results output at each stage are as follows Figures 6 to 8 The TTP value data of the 96-well plate well position distribution obtained after processing and analyzing according to steps a to i are shown in Table 1.

[0169] Table 1: 96-well plate well position distribution TTP value data table

[0170]

[0171] Note: PC means positive; NC means negative.

[0172] Based on the above results, it can be calculated that the average TTP values ​​of the simulated samples of 4 low-concentration nucleic acid templates of 500, 300, 200, and 100 copies / mL, as well as 2 positive quality controls and 2 negative quality controls are 14.64, 14.69, 16.11, 17.03, 10.17, 9.54, Inf, and Inf, respectively, and the corresponding coefficients of variation of TTP values ​​within the group are 22.16%, 13.91%, 28.30%, 38.83%, 3.06%, 3.41%, 0, and 0, respectively.

[0173] Example 2: Comparison of real-time signal result determination analysis method based on dual-channel loop-mediated isothermal amplification and endpoint method

[0174] 1) LAMP real-time signal data collection

[0175] According to an embodiment of the present invention, in order to verify the feasibility of the above-mentioned real-time signal result determination method for dual-channel signal processing analysis, this embodiment adopts a loop-mediated isothermal amplification (LAMP) experimental method, and prepares a single-channel detection of a nucleic acid template containing several different concentrations of a double-stranded probe system developed by MGI (MGI), and the total volume of the detection system is 30 μl, which contains 12 μl of template. The detection system contains two different fluorescence wavelength probes modified by FAM and ROX, respectively. The amplification container is a 96-well PCR plate, which contains 10 positive quality controls with 5 different conditions (2 types of FAM-modified fluorescent probes O-Q2 and O-Q1, 3 types of ROX-modified fluorescent probes YN-N3, YN, N3, each with 2 replicates), 10 negative quality controls with 5 different conditions (2 types of FAM-modified fluorescent probes O-Q2 and O-Q1, 3 types of ROX-modified fluorescent probes YN-N3, YN, N3, each with 2 replicates), 76 low-concentration nucleic acid template test samples with 5 different conditions (2 types of FAM-modified fluorescent probes O-Q2 and O-Q1, 3 types of ROX-modified fluorescent probes YN-N3, YN, N3, each with 8 replicates, each with 20 replicates), and the sample concentration of the preservation solution before extraction of the test samples is 500 copies / ml. The real-time signal acquisition device is Hangzhou Biori FQD-96A, running the device client software Gene-9660, and setting the acquisition program to 25 time points in three stages: pre-acquisition, reverse transcription, and isothermal amplification. The fluorescence signals of the two channels of FAM and ROX are collected at the same time. The temperature is set to 30°C during signal acquisition, and the temperature is set to 65°C during reverse transcription and isothermal amplification. Click "Start Run" to start the acquisition program. Until the acquisition program completes 25 acquisition actions and obtains all amplified real-time signal data, the client software Gene-9660 will automatically draw the obtained data synchronously. Fig. 9Since the client software Gene-9660 does not have the function of analyzing LAMP real-time signals, the software can be used to export the raw data of LAMP real-time signals to Excel or text format files and save them for data analysis.

[0176] 2) LAMP real-time signal data analysis

[0177] Based on the technical solution disclosed in the present invention, the LAMP real-time signal raw data is processed and analyzed. The LAMP real-time signal raw data is read, and the real-time signal raw data is divided into two corresponding independent data tables according to the dye channel type (FAM or ROX) and saved. The above two independent data tables are read respectively, and the processing and analysis of the real-time signal raw data of the FAM channel and the ROX channel are completed, and the order is not limited. The specific steps are as follows:

[0178] a. Generate a two-dimensional matrix of real-time signal raw data according to the signal acquisition time point and the amplified real-time signal value (the matrix contains 96×25 real-time signal data);

[0179] b. Take the real-time signal values ​​of the first six signal acquisition time points to calculate the signal threshold of each amplifier (a total of 96). The calculation steps are as follows:

[0180] First, the average value (AVG) and standard deviation (STD) of the real-time signal values ​​of the first six signal acquisition time points of each amplifier were calculated;

[0181] Secondly, the signal threshold of each amplifier is calculated based on the formula (S_thresh = AVG + N1 × STD, N1 is 10);

[0182] c. Calculate the maximum value of the last 19 real-time signal values ​​of each amplifier (25 minus the first 6 signal values), compare the value with the corresponding signal threshold S_thresh, and record the threshold comparison result. If it is greater than the threshold, it is recorded as 1, and if it is less than the threshold, it is recorded as 0. According to the threshold comparison result, the amplifier with a value of 1 is preliminarily determined as positive (amplified), and the amplifier with a value of 0 is preliminarily determined as negative (not amplified);

[0183] d. The corresponding real-time signal values ​​of all negative amplifiers are fitted with a polynomial (the polynomial order is set to 8) to calculate the average baseline of the unamplified real-time signal data. Based on the above average baseline data, the two-dimensional matrix of the real-time signal raw data is processed to obtain a two-dimensional matrix of the real-time signal data after baseline correction (the matrix contains 96×25 real-time signal data);

[0184] e. Initialize the two-dimensional matrix of real-time signal data after baseline correction according to the real-time signal value at the first signal acquisition time point (data alignment), and normalize all signal data values ​​of the two-dimensional matrix to [0, 1]. Obtain the initial normalized two-dimensional matrix of real-time signal data;

[0185] f. According to the two-dimensional matrix of initial normalized real-time signal data, take the initial normalized real-time signal values ​​of the first 6 signal acquisition time points to calculate the initial normalized signal threshold of each amplifier (a total of 96). The calculation steps are as follows:

[0186] First, the average of the initial normalized real-time signal values ​​(AVG) of the first six signal acquisition time points of each amplifier was calculated. norm ) and standard deviation (STD norm );

[0187] Secondly, based on the formula (S norm _thresh=AVG norm +N2×STD norm , N2 is 10) Calculate the initial normalized signal threshold S of each amplifier norm _thresh;

[0188] g. Calculate the maximum value of the last 19 initial normalized real-time signal values ​​of each amplifier (25 minus the first 6 signal values), and compare it with the corresponding initial normalized signal threshold S norm _thresh compares the size and records the threshold comparison result. If it is greater than the threshold, it is recorded as 1, and if it is less than the threshold, it is recorded as 0. According to the threshold comparison result, the amplifier with a value of 1 is initially judged as positive (amplified), and the amplifier with a value of 0 is initially judged as negative (not amplified);

[0189] h. Use the smoothing spline fitting method to fit the initial normalized real-time signal data of all amplifiers to obtain the real-time signal smooth curve corresponding to each amplifier, determine the unique intersection of the curve and the corresponding initial normalized signal threshold, and calculate the signal acquisition time value corresponding to the intersection. This time value is the positive reporting time TTP value of the amplifier. Since there is no intersection between the real-time signal smooth curve of each negative amplifier and the corresponding threshold, its positive reporting time TTP value is set to infinity (Inf);

[0190] i. Finally, the real-time signal curve processing results output at each stage are as follows Fig.10 The TTP value data of the 96-well plate well position distribution obtained after processing and analyzing according to steps 1 to 8 are shown in Table 2.

[0191] Table 2: 96-well plate well position distribution TTP value data table

[0192]

[0193] Based on the above results, it can be calculated that the average TTP values ​​of the simulated samples of five low-concentration nucleic acid templates, YN-N3, YN, N3, O-Q2, and O-Q1, as well as the corresponding positive quality controls and negative quality controls are 8.35, 9.80, 13.21, 7.86, 6.91, 7.13, 7.32, 7.56, 5.46, 5.37, Inf, Inf, 23.37, Inf, and 18.35, respectively. The corresponding coefficients of variation of TTP values ​​within the group are 27.55%, 12.23%, 34.44%, 31.72%, 13.65%, 0.99%, 0.19%, 6.36%, 1.43%, 0%, 0%, 0%, 4.15%, 0%, and 5.90%, respectively.

[0194] By comparison Fig.11 Compared with the TTP value results in Table 2, it can be found that the fluorescence signal image of the 96-well PCR plate endpoint method has a corresponding relationship with the values ​​in Table 2. This further verifies the feasibility of the real-time signal result determination and analysis method based on dual-channel loop-mediated isothermal amplification.

[0195] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0196] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A method for classifying amplification containers, characterized in that: include: For each of the multiple amplification containers, respectively, signal acquisition is performed at multiple given time points to obtain an original real-time signal data set; Based on the original real-time signal data set, classify at least one of the plurality of amplification containers once to obtain a preliminarily determined amplification container and a preliminarily determined non-amplification container; Determine an amplification baseline based on at least a portion of the signal of the initially determined non-amplification container, and use the amplification baseline to correct at least a portion of the original real-time signal data set to obtain a corrected real-time signal data set; Based on the corrected real-time signal data set, secondary classification is performed on at least one of the plurality of amplification containers to obtain a final judgment amplification container and a final judgment non-amplification container.

2. The method according to claim 1, characterized in that For a given amplification container, the primary classification is performed by: Determining a first signal threshold for said amplification based on the signal at at least one initial time point; and According to a first difference between the signal of the amplification container at another time point and the first signal threshold, the amplification container is classified into an amplification container and a non-amplification container. For a given amplification container, the secondary sorting is performed by the following steps: determining a second signal threshold based on the corrected signal at the initial at least one time point; and performing a secondary classification of the amplification container between an amplification container and a non-amplification container according to a second difference between the signal of the amplification container at another time point and the second signal threshold; Optionally, the amplification baseline can be obtained by calculation methods such as fitting, interpolation, regression or averaging.

3. The method according to claim 2, characterized in that The first signal threshold S_thresh is obtained by the following calculation formula: S_thresh=AVG+N1×STD Among them, AVG represents the average value of the original real-time signal data selected for a classification; STD represents the standard deviation of the original real-time signal data selected for a classification; N1 is an integer.

4. The method according to claim 2 or 3, characterized in that: The first difference is determined by comparing the signal of the amplification container at other time points with the first signal threshold; Optionally, the signal at the other time point is greater than the first signal threshold, which is an indication that the amplification container is a preliminary amplification container; The signal at least one other time point is less than the first signal threshold, which is an indication that the amplification container is a preliminarily judged non-amplification container.

5. The method according to claim 2, characterized in that: The second signal threshold S norm _thresh is obtained by the following calculation formula: S norm _thresh=AVG norm +N2×STD norm Among them, AVG norm Indicates the average value of the corrected real-time signal data selected for secondary classification; STD norm Represents the standard deviation of the corrected real-time signal data selected for secondary classification; N2 is an integer.

6. The method according to claim 2 or 5, characterized in that: The second difference is determined by comparing the signal of the amplification container at other time points with the second signal threshold; Optionally, the signal at the other time point is greater than the second signal threshold, which is an indication that the amplification container is a final judgment amplification container; The signal at the other time point is less than the second signal threshold, which is an indication that the amplification container is a final judgment non-amplification container.

7. A method for determining the positive time of an amplification reaction, characterized in that: include: performing an amplification reaction in a plurality of amplification vessels; According to the method of claim 1, the plurality of amplification containers are classified to determine final judgment amplification containers and final judgment non-amplification containers; constructing a data function of the corrected real-time signal with respect to sampling time based on at least a portion of the plurality of amplification vessels; Determining a signal threshold of the final judgment non-amplification container based on the corrected real-time signal of the final judgment non-amplification container; and Based on the data function, determining the positive reporting time of a given amplification container corresponding to the signal threshold; Optionally, the data function can be calculated by polynomial fitting, spline fitting or exponential fitting method.

8. A method for analyzing a biological sample by an amplification reaction, characterized in that: include: Obtaining biological samples suspected of containing nucleic acid; For the biological sample, performing an amplification reaction in a plurality of amplification containers; The method of claim 7, determining a positive reporting time for a given amplification container; and Based on the positive reporting time, the nucleic acid content in the biological sample is analyzed.

9. A method for determining the nucleic acid content in a nucleic acid sample, characterized in that: include: Performing an amplification reaction on the nucleic acid sample in a plurality of amplification containers; The method of claim 7, determining a positive reporting time for a given amplification container; and Based on the positive reporting time, the content of nucleic acid in the nucleic acid sample is determined.

10. The method according to any one of claims 1 or 7 to 9, characterized in that: The amplification is selected from isothermal amplification; Optionally, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling circle amplification, strand displacement amplification, multiple displacement amplification, transcription-mediated amplification, single primer amplification and helicase-dependent amplification.

11. A device for sorting amplification containers, characterized in that: include: A data collection module, used for collecting signals at a plurality of given time points for each of the plurality of amplification containers, so as to obtain an original real-time signal data set; A first classification determination module, the first classification predetermined module is connected to the data collection module, and is used to classify at least one of the plurality of amplification containers based on the original real-time signal data set, so as to obtain a preliminarily determined amplification container and a preliminarily determined non-amplification container; a correction module, the correction module being connected to the first classification determination module, and being used to determine an amplification baseline based on at least a portion of the signal of the initially determined non-amplification container, and to use the amplification baseline to correct at least a portion of the original real-time signal data set, so as to obtain a corrected real-time signal data set; A second classification and determination module is connected to the correction module and is used to perform secondary classification on at least one of the multiple amplification containers based on the corrected real-time signal data set, so as to obtain a final judgment amplification container and a final judgment non-amplification container.

12. A system for determining the positive time of an amplification reaction, characterized in that: include: an amplification reaction device, wherein the amplification reaction device is used to perform an amplification reaction; and The device of claim 11, connected to the plurality of amplification containers, for sorting the amplification containers; and A data function construction device, connected to the device of claim 11, for constructing a data function of the corrected real-time signal relative to the sampling time based on at least a portion of the plurality of amplification containers; and A signal threshold determination device for a final judgment non-amplification container, the signal threshold determination device for a final judgment non-amplification container is connected to the data function construction device, and is used to determine the signal threshold of the final judgment non-amplification container based on the corrected real-time signal of the final judgment non-amplification container; and A positive reporting time acquisition device is connected to the signal threshold determination device and the data function construction device for the final judgment non-amplification container, and is used to determine the positive reporting time of a given amplification container corresponding to the signal threshold based on the data function.

13. A system for analyzing biological samples by amplification reaction, characterized in that: include: A biological sample acquisition device, wherein the biological sample acquisition device is used to acquire a biological sample suspected of containing nucleic acid; and The system for determining the positive reporting time of an amplification reaction as claimed in claim 12, wherein the system is connected to the biological sample acquisition device and is used to determine the positive reporting time of a given amplification container; and An analysis device is connected to the system for determining the positive time of the amplification reaction and is used to analyze the nucleic acid content in the biological sample.

14. A system for determining the nucleic acid content in a nucleic acid sample, characterized in that: include: The system for determining the positive reporting time of an amplification reaction as claimed in claim 12 is used to determine the positive reporting time of a given amplification container; and A nucleic acid content acquisition device is connected to the system and is used to determine the nucleic acid content in the nucleic acid sample.

15. A computer program product, characterized in that The computer program product comprises computer instructions, and when part or all of the computer instructions are run on a computer, the method according to any one of claims 1 to 10 is executed.

16. A computing device, characterized in that: include: Processor and memory; The memory is used to store computer programs; The processor is configured to execute the computer program to implement the method according to any one of claims 1 to 10.

17. A computer-readable storage medium, characterized in that: The storage medium includes computer instructions. When the instructions are executed by a computer, the computer implements the method according to any one of claims 1 to 10.

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