Method and device suitable for classification of sparse signal amplification containers

Abstract

The present invention relates to a method and device suitable for classification of sparse signal amplification containers. The method comprises: for each of a plurality of amplification containers, performing signal acquisition at a given time point to obtain an original real-time signal data set; on the basis of the original real-time signal data set, performing primary classification on at least one of the plurality of amplification containers to obtain an initially determined amplification container and an initially determined non-amplification container; determining an amplification baseline on the basis of at least some of signals of the initially determined non-amplification container, and using the amplification baseline to correct at least a part of the original real-time signal data set to obtain a corrected real-time signal data set; and on the basis of the corrected real-time signal data set, performing secondary classification on at least one of the plurality of amplification containers to obtain a finally determined amplification container and a finally determined non-amplification container.

Description

Method and device for classifying sparse signal amplification containers Technical Field

[0001] The present invention relates to the fields of bioinformatics analysis and molecular biology detection, and in particular to a method and device suitable for classifying sparse signal 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 the qualitative and quantitative detection of biochemical analytes such as nucleic acids. PCR uses variable temperature cycles to allow the double-stranded DNA 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] Over the past 20 years, isothermal nucleic acid amplification (NAA) has become an important diagnostic tool, not only for clinical applications but also for food quality control and environmental monitoring. Nucleic acid isothermal amplification (NAA) is a new solution proposed to overcome the various limitations and shortcomings of PCR technology. It can rapidly amplify the copy number of target DNA or RNA fragments at a constant temperature. This type of technology can reduce the complex steps of sample and reagent preparation, completely eliminating the reliance on thermal cyclers, and can greatly simplify the complexity of nucleic acid detection protocols, which is of great significance for applications in on-site point-of-care testing. 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 rely on temperature cycling to alter the conformation of the template strand. Most utilize a polymerase-mediated strand displacement mechanism, distinct from the thermal denaturation used in PCR, to achieve template strand release and primer annealing. The polymerase-mediated strand displacement reaction (SDR) is a widely used technique in nucleic acid amplification reactions and a mimic of the in vivo nucleic acid replication process. This process primarily involves primer extension by a polymerase with strand-displacing activity and the displacement of the downstream strand by the resulting new strand. Polymerase-mediated strand displacement leads to unwinding of the original duplex and generation of a new duplex, resulting in amplification. This displacement reaction replaces the high-temperature unwinding required in traditional temperature-dependent reactions, can be performed over a wide temperature range, and is simple and convenient to operate. In recent years, SDR has been widely used in various fields of molecular biology due to its high specificity and sensitivity. It has also garnered significant attention in signal amplification and diagnostic biosensing applications.

[0005] Based on the above principles, by designing and constructing templates, primers, or intermediates with specific structures, it is possible to achieve cyclic reuse of templates and intermediates or parallel extension of primers. This self-circulating chain displacement amplification mechanism often produces multiple products through repeated amplification of a single template, achieving faster product synthesis and significantly improving amplification efficiency and sensitivity. Self-circulating chain displacement amplification includes LAMP, RCA, MDA, SDA, SPIA, and other technologies. Among them, LAMP, the most widely used technology, has the typical characteristics and advantages of self-circulating chain displacement amplification, and also has the highest amplification efficiency and sensitivity.

[0006] Therefore, there is an urgent need in this field to develop a universal method for analyzing sparse real-time signal results suitable for isothermal nucleic acid amplification.

[0007] Summary of the Invention

[0008] This application is filed by the inventor based on the following findings:

[0009] 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 their respective collected signals, and the real-time detection results cannot be compared in parallel. In addition, the real-time detection procedures of various technical solutions with the same labeling method are artificially preset, and the preset reading number and reading time interval are relatively arbitrary, and there is a lack of unified standards, 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.

[0010] Secondly, the calculation methods for the quantitative index values ​​used in some technical solutions lack a unified standard, and are often processed based on the default analysis methods of the real-time PCR software program (the algorithm is based on the PCR exponential kinetic model), which is not necessarily compatible with and applicable to isothermal amplification (super-exponential kinetic model). 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. This makes it impossible to achieve semi-quantitative or quantitative comparison of test results, and it cannot meet the automation and throughput requirements of nucleic acid testing.

[0011] More importantly, because the real-time fluorescence thermal cycle or real-time fluorescence constant temperature device needs to spend more running time each time when reading or collecting each channel signal. In order to better meet the needs of rapid response, it is necessary to further compress the running time of the nucleic acid amplification real-time detection technical solution. The commonly used strategy is to cut the number or frequency of real-time signal reading or collection as much as possible (the extreme case is to perform 1 signal reading / collection before and after the amplification reaction is started, that is, degenerate into the end point detection technical solution). However, the reduction in the number or frequency of real-time signal reading / collection will synchronously bring about the loss of amplification reaction kinetic information, and the subtle change process of the real-time signal cannot be accurately portrayed. For this reason, the inventor has designed a nucleic acid amplification real-time signal result determination method to solve the problems such as the poor comparability of the result determination caused by the sparse real-time signal.

[0012] In the first aspect of the present invention, the present invention proposes a method for classifying sparse signal amplification containers. According to an embodiment of the present invention, the method comprises: for each of a plurality of amplification containers, respectively, performing signal acquisition at a given time point to obtain an original real-time signal data set; based on the original real-time signal data set, performing a classification for at least one of the plurality of amplification containers 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, determining an amplification baseline, and using the amplification baseline to correct at least a portion of the amplification real-time signal data set to obtain a corrected real-time signal data set; based on the corrected real-time signal data set, performing a secondary classification for at least one of the plurality of amplification containers to obtain a final judgment amplification container and a final judgment non-amplification container. According to an embodiment of the present invention, the method simplifies the existing processing method of amplification real-time detection signals, and uses existing biochemical experimental data to perform algorithm optimization and iteration, improves calculation accuracy, data processing efficiency and versatility of analysis methods, is more suitable for the result determination analysis of sparse real-time signals, realizes a rapid and automated analysis method for determining nucleic acid detection results in various types of amplification containers (such as 96-well PCR plates), and forms a technical standard.

[0013] In some examples of the present application, the amplification baseline can be obtained by calculation methods such as fitting, interpolation, regression or averaging.

[0014] In some examples of the present application, the number of given time points is not less than 3. The number of given time points is selected from the minimum number of time points that does not affect the analysis result.

[0015] In some examples of the present application, the amplification process includes calculating continuous real-time signal curve data corresponding to each amplification container or adding time point real-time signal data of the amplification container.

[0016] The calculation of continuous real-time signal curve data corresponding to each amplification container is performed by continuously monitoring the fluorescence signal during the amplification reaction to obtain a real-time fluorescence intensity curve. On this curve, key amplification characteristic points (such as the starting point, exponential growth period, plateau period, etc.) can be identified, thereby calculating the quality parameters of the amplification reaction (such as the positive reporting time and amplification efficiency, etc.).

[0017] According to an embodiment of the present invention, the method for classifying sparse signal 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 primary classification is performed by: determining a first signal threshold for the amplification based on a signal at at least one initial time point; and performing a primary classification of the amplification container between an amplification container and a non-amplification container based on a first difference between the signal at other time points of the amplification container and the first signal threshold; for a given amplification container, the secondary classification is performed by: determining a second signal threshold based on a corrected signal at at least one initial time point; and performing a secondary classification of the amplification container between an amplification container and a non-amplification container based on a second difference between the signal at other time points of the amplification container and the second signal threshold. The use of the two-classification method can analyze signal data at different levels, improve the accuracy and stability of the 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 strip, a 96-well 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-tube strip, 96- or 384-well PCR plate, droplet and microfluidic amplification pool. In this 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 first signal threshold S_thresh is obtained by the following calculation formula: S_thresh=AVG+N1×STD

[0022] Wherein, AVG represents the average value of the amplified real-time signal data selected for a classification; STD represents the standard deviation of the amplified real-time signal data selected for a classification; and N1 is an integer.

[0023] In some examples of the present application, in the aforementioned formula for calculating the first signal threshold, N1 takes values ​​ranging from 1 to 25. Specifically, N1 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, and 25. In some preferred embodiments of the present application, N1 takes a value of 10.

[0024] 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.

[0025] 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.

[0026] According to an embodiment of the present invention, if the signal at other time points is greater than the first signal threshold, it indicates that the amplification container is a preliminarily judged amplification container; if the signal at other time points is less than the first signal threshold, it indicates that the amplification container is a preliminarily judged non-amplification container.

[0027] It should be noted that the initial judgment of amplification (initial judgment of positive amplification) in this 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 an 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.

[0028] According to an embodiment of the present invention, the second signal threshold S norm _thresh is obtained by the following calculation formula: S norm _thresh=AVG norm +N2×STD norm

[0029] 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.

[0030] In some examples of the present application, in the aforementioned second signal threshold calculation formula, N2 takes values ​​ranging from 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, and 25. In some preferred embodiments of the present application, N2 takes a value of 10.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] According to an embodiment of the present invention, if the signal at other time points is greater than the second signal threshold, it indicates that the amplification container is a final-judgment amplification container; if the signal at other time points is less than the second signal threshold, it indicates that the amplification container is a final-judgment non-amplification container.

[0035] It should be noted that the final judgment of amplification (final judgment of positive amplification) described in this 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.

[0036] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0037] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.

[0038] According to an embodiment of the present invention, prior to the primary classification, the process further includes performing expansion processing on the original real-time signal data set. This step increases the density of the original real-time signal data, i.e., obtaining more data points at a specific time point. This increased data density is beneficial in providing more accurate and detailed signal information, thereby making subsequent classification, analysis, and prediction more accurate.

[0039] According to an embodiment of the present invention, the expansion processing is achieved by performing data processing on the data matrix of the original real-time signal data set; the data processing is performed using at least one of fitting, interpolation, regression, or averaging methods. Data expansion processing using methods such as fitting, interpolation, regression, or averaging can fill gaps in the signal data, making the real-time signal data more continuous and substantial.

[0040] In one example of the present application, a data matrix of an original real-time signal data set is read to obtain T real-time signal data from an amplifier. Then, using any of fitting, interpolation, regression, or averaging methods, the obtained T real-time signal data are expanded into a set of real-time signal data with a higher data density (e.g., T+E), where E is a positive integer.

[0041] In a second aspect of the present invention, the present invention proposes a method for determining the time for reporting a positive amplification reaction (TTP value). According to an embodiment of the present invention, the method includes: performing an amplification reaction in a plurality of amplification containers; and classifying the plurality of amplification containers according to the first aspect of the present application or the method described in any embodiment, determining the final judgment amplification container and the final judgment non-amplification container; and 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 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 reporting 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 calculation accuracy and experimental efficiency of the isothermal amplification reaction reporting time (TTP value) through an automated and efficient data processing scheme, while also reducing resource waste.

[0042] In some examples of this 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 the 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 strip, a 96-well 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-tube strip, 96- or 384-well PCR plate, droplet and microfluidic amplification pool. In this 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 amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal 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 isothermal amplification reaction in multiple isothermal amplification containers for the biological sample; determining the positive reporting time of a given amplification container according to the method described in the second aspect; 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 analysis 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 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 through 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 strip, a 96-well 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-tube strip, 96- or 384-well PCR plate, droplet and microfluidic amplification pool. In this 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 amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal 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 isothermal amplification reaction on the nucleic acid sample in a plurality of 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 nucleic acid content 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 detection results obtained based on different labeling methods; it is also suitable for analyzing the result judgment limitations 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 strip, a 96-well 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-tube strip, 96- or 384-well PCR plate, droplet and microfluidic amplification pool. In this 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 amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.

[0062] In a fifth aspect, 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: performing an isothermal amplification reaction in multiple isothermal amplification vessels using primers specific for the target nucleic acid molecule; determining a positive detection time for a given amplification vessel 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 detection time.

[0063] According to an embodiment of the present invention, the method is applicable to low-quality and low-concentration nucleic acid samples and does not require specialized instruments and complex operating steps, making the detection process simpler and more efficient. In addition, the use of multiple amplification containers for simultaneous amplification reactions can greatly improve the efficiency and accuracy of detection. The content of the target nucleic acid molecule is determined based on the positive reporting time, thereby avoiding the need for accurate quantitative thermal cycling amplification reactions in traditional PCR methods, thereby reducing the difficulty and time cost of experimental operations.

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

[0065] In some examples of the present application, the method can quickly calculate the expression level of the target gene in the test nucleic acid sample based on the positive reporting time.

[0066] 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 strip, a 96-well or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0067] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-tube strip, 96- or 384-well PCR plate, droplet and microfluidic amplification pool. In this application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0068] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.

[0069] In a sixth aspect, the present invention provides a device suitable for classifying sparse signal amplification containers. According to an embodiment of the present invention, the device includes: a data collection unit for collecting signals from each of a plurality of amplification containers at a plurality of given time points to obtain an original real-time signal data set; a data expansion unit, connected to the data collection unit, for performing expansion processing on the original data to obtain an amplified real-time signal data set; a first classification determination unit, connected to the data collection module, for performing a primary classification on at least one of the plurality of amplification containers based on the amplified real-time signal data set to obtain a preliminary judgment of an amplification container and a preliminary judgment of a non-amplification container; a correction unit, connected to the first classification determination module, for determining an amplification baseline based on at least a portion of the signals from the preliminary judgment of a non-amplification container, and correcting at least a portion of the amplified real-time signal data set using the amplification baseline to obtain a corrected real-time signal data set; and a second classification determination unit, connected to the correction module, for performing a secondary classification on at least one of the plurality of amplification containers based on the corrected real-time signal data set to obtain a final judgment of an amplification container and a final judgment of a non-amplification container. According to embodiments of the present invention, the device can automatically collect time-point signal data from a sample in an amplification container, eliminating the need for manual intervention and improving experimental efficiency. Furthermore, even when the sample receives less real-time signal, accurate analysis can still be performed. Furthermore, the dual classification process of positive and negative amplification improves classification accuracy. Because the device is based on machine learning technology, it can also self-learn and optimize, adapting to amplification container classification tasks under different conditions.

[0070] It should be noted that, as shown in Figure 1, from a structural point of view, the data collection unit S001 is connected to the data expansion unit S002, the data expansion unit S002 is connected to the first classification judgment unit S003, the first classification judgment unit S003 is connected to the correction unit S004, and the correction unit S004 is connected to the second classification judgment unit S005.

[0071] According to an embodiment of the present invention, the device suitable for classifying sparse signal amplification containers may further include at least one of the following technical features:

[0072] According to an embodiment of the present invention, for a given amplification container, the primary classification is performed by: determining a first signal threshold for the amplification based on a signal at at least one initial time point; and performing a primary classification of the amplification container between an amplification container and a non-amplification container based on a first difference between the signal at other time points of the amplification container and the first signal threshold; for a given amplification container, the secondary classification is performed by: determining a second signal threshold based on a corrected signal at at least one initial time point; and performing a secondary classification of the amplification container between an amplification container and a non-amplification container based on a second difference between the signal at other time points of the amplification container and the second signal threshold. The use of the two-classification method can analyze signal data at different levels, improve the accuracy and stability of the classification, reduce the error rate, and make the final classification result more reliable and accurate.

[0073] 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 strip, a 96-well or 384-well PCR plate, a droplet, and a microfluidic amplification pool.

[0074] It should be noted that the amplification container is not limited to the aforementioned single PCR tube, 8-tube strip, 96- or 384-well PCR plate, droplet and microfluidic amplification pool. In this application, any container that can achieve nucleic acid amplification can be used as an amplification container.

[0075] According to an embodiment of the present invention, the first signal threshold S_thresh is obtained by the following calculation formula: S_thresh=AVG+N1×STD

[0076] Wherein, AVG represents the average value of the amplified real-time signal data selected for a classification; STD represents the standard deviation of the amplified real-time signal data selected for a classification; and N1 is an integer.

[0077] In some examples of the present application, in the aforementioned formula for calculating the first signal threshold, N1 is an integer ranging from 1 to 25. Specifically, N1 is an integer ranging from 1 to 25. Specifically, N1 is an integer ranging from 1 to 25. In some preferred embodiments of the present application, N1 is 10.

[0078] 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.

[0079] 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.

[0080] According to an embodiment of the present invention, if the signal at other time points is greater than the first signal threshold, it indicates that the amplification container is a preliminarily judged amplification container; if the signal at other time points is less than the first signal threshold, it indicates that the amplification container is a preliminarily judged non-amplification container.

[0081] It should be noted that the initial judgment of amplification (initial judgment of positive amplification) in this 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 an 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.

[0082] According to an embodiment of the present invention, the second signal threshold S norm _thresh is obtained by the following calculation formula: S norm _thresh=AVG norm +N2×STD norm

[0083] 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.

[0084] In some examples of the present application, in the aforementioned second signal threshold calculation formula, N2 takes values ​​ranging from 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, and 25. In some preferred embodiments of the present application, N2 takes a value of 10.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] According to an embodiment of the present invention, if the signal at other time points is greater than the second signal threshold, it indicates that the amplification container is a final-judgment amplification container; if the signal at other time points is less than the second signal threshold, it indicates that the amplification container is a final-judgment non-amplification container.

[0089] It should be noted that the final judgment of amplification (final judgment of positive amplification) described in this 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.

[0090] According to an embodiment of the present invention, the amplification is selected from isothermal amplification.

[0091] According to an embodiment of the present invention, the isothermal amplification is selected from at least one of loop-mediated amplification, rolling amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.

[0092] In the seventh aspect of the present invention, the present invention proposes a system for determining the time of reporting a positive 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 of the present invention, the device is connected to the multiple amplification containers for classifying the amplification containers; and a data function construction device, the data function construction device is connected to the device described in the sixth aspect of the present invention, for constructing a data function of the corrected real-time signal relative to the sampling time based on at least a portion 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, for determining 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, for determining the reporting positive time of a given amplification container corresponding to the signal threshold based on the data function.

[0093] According to an embodiment of the present invention, the system for determining the positive reporting time of the amplification reaction can accurately judge positive samples and negative samples, and based on the obtained positive reporting time data, parameters such as sensitivity, specificity, precision, repeatability, reproducibility, and detection limit can be evaluated, 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, as shown in Figure 2, from a structural point of view, the amplification reaction device S100 is connected to the device S200 suitable for classifying sparse signal amplification containers (including: data collection unit S001, data expansion unit S002, first classification judgment unit S003, correction unit S004 and second classification judgment unit S005), the device S200 suitable for classifying sparse signal amplification containers is connected to the data function construction device S300, the data function construction device S300 is connected to the signal threshold determination device S400 for the final judgment non-amplification container, and the signal threshold determination device S400 for the final judgment non-amplification container is connected to the positive reporting time acquisition device S500.

[0095] In its eighth aspect, the present invention provides a system for analyzing a biological sample through an amplification reaction. According to an embodiment of the present invention, the system includes: a biological sample acquisition device, configured to acquire a biological sample suspected of containing nucleic acid; the system for determining the time of positive amplification reaction detection as described in the seventh aspect of the present invention, connected to the biological sample acquisition device and configured to determine the time of positive amplification reaction detection for a given amplification container; and an analysis device, connected to the system for determining the time of positive amplification reaction detection, configured 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 through amplification reaction is that it avoids the complex data analysis process in traditional PCR technology. This model structure can analyze biological samples quickly, accurately and automatically.

[0097] It should be noted that, as shown in Figure 3, from a structural point of view, the biological sample acquisition device S600 is connected to the system X01 for determining the time when the amplification reaction is reported as positive (including: an amplification reaction device S100, a device S200 suitable for classifying sparse signal amplification containers, a data function construction device S300, a signal threshold determination device S400 for final judgment of non-amplification containers, and a positive reporting time acquisition device S500), and the system X01 for determining the time when the amplification reaction is reported as positive is connected to the analysis device S700.

[0098] In a ninth aspect, 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 time when a given amplification reaction reports a positive result as described in the seventh aspect of the present invention, configured to determine the time when a given amplification container reports a positive result; and a nucleic acid content acquisition device connected to the system for determining 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 the amplification reaction, avoiding the tedious data analysis process and saving time and cost.

[0100] It should be noted that, as shown in Figure 4, from a structural point of view, the system X01 for determining the positive reporting time of the amplification reaction (including: an amplification reaction device S100, a device suitable for classifying sparse signal amplification containers S200, a data function construction device S300, a signal threshold determination device S400 for final judgment of non-amplification containers, and a positive reporting time acquisition device S500) is connected to the nucleic acid content acquisition device S800.

[0101] In its tenth aspect, the present invention provides 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, the system comprises: the system for determining the positive amplification reaction reporting time described in the seventh aspect of the present invention, configured to determine the positive amplification reaction reporting time of a given amplification container; and a target nucleic acid molecular weight acquisition device, connected to the system, configured 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 is suitable for detecting low-quality and low-concentration nucleic acid samples; in addition, the use of multiple amplification containers to perform amplification reactions simultaneously can greatly improve the efficiency and accuracy of detection; and the content of the target nucleic acid molecule is determined based on the positive reporting time, avoiding the need for accurate quantitative thermal cycling amplification reactions in traditional PCR methods, thereby reducing the difficulty and time cost of experimental operations; the system automatically determines the content of target nucleic acid molecules in nucleic acid samples (without manual intervention).

[0103] It should be noted that, as shown in Figure 5, from a structural point of view, the system X01 for determining the positive reporting time of the amplification reaction (including: an amplification reaction device S100, a device suitable for classifying sparse signal amplification containers S200, a data function construction device S300, a signal threshold determination device S400 for final judgment of non-amplification containers, and a positive reporting time acquisition device S500) is connected to the target nucleic acid molecular weight acquisition device S900.

[0104] In an eleventh aspect, 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 executed on a computer, the method described in the first, second, third, fourth, or fifth aspect of the present invention is executed.

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

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

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

[0108] It should be noted that various embodiments of the systems and techniques described herein 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 embodiments may include being implemented in one or more computer programs that are executable and / or interpreted on a programmable system comprising at least one programmable processor, which may be a special-purpose or general-purpose programmable processor that can 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. Such program code 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 when the program code is executed by the processor or controller, the functions / operations specified in the flow chart and / or block diagram are implemented. The program code can be executed entirely on the machine, partially on the machine, as a stand-alone software package, partially on the machine and partially on a remote machine, or entirely on a remote machine or server.

[0110] In the context disclosed herein, 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 apparatus. 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 apparatus, or any suitable combination of the foregoing. More specific examples of machine-readable storage media may include an electrical connection based on one or more wires, a portable computer disk, a hard disk, RAM, ROM, EPROM (Electrically Programmable Read-Only-Memory) or flash memory, optical fiber, CD-ROM (Compact Disc Read-Only Memory), an optical storage device, a magnetic storage device, 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 can be implemented in a computing system that includes back-end 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 front-end components (e.g., a user computer with a graphical user interface or 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 back-end components, middleware components, or front-end components. The components of the system can 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 server are generally remote from each other and typically interact via a communication network. This client-server relationship is established by computer programs running on the respective computers, establishing a client-server relationship. The server may be a cloud server, also known as a cloud computing server or cloud host, a host product within the cloud computing service ecosystem that addresses the management difficulties and limited scalability of traditional physical hosts and VPS services ("Virtual Private Servers" or simply "VPS"). The server may also be a server in a distributed system or a server integrated with blockchain.

[0114] It should be understood that the various forms of the processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this disclosure can be performed in parallel, sequentially, or in a different order, as long as the desired results of the technical solutions disclosed in this disclosure can be achieved. This is not limited herein.

[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 set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by 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 readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0118] FIG1 is a schematic diagram of a device structure suitable for sparse signal amplification container classification according to one embodiment of the present invention;

[0119] FIG2 is a schematic diagram of a system structure for determining the positive time of an amplification reaction according to one embodiment of the present invention;

[0120] FIG3 is a schematic diagram of a system structure for analyzing biological samples through an amplification reaction according to one embodiment of the present invention;

[0121] FIG4 is a schematic diagram of the structure of a system for determining the nucleic acid content in a nucleic acid sample according to one embodiment of the present invention;

[0122] FIG5 is a schematic diagram of a system structure for determining the content of target nucleic acid molecules in a nucleic acid sample according to one embodiment of the present invention;

[0123] FIG6 is a real-time signal curve of a single-channel LAMP according to Example 1 of the present invention (raw data of Biori FQD-96A);

[0124] Figure 7 is a schematic diagram of the real-time signal curve processing results in Example 1 of the present invention; wherein: (A) original signal curve; (B) step c, positive original signal curve; (C) step d, negative original signal curve; (D) step e, polynomial fitting average baseline graph; (E) step e, correction and initialization signal curve graph. (F) steps e and h, normalized signal curve spline fitting graph;

[0125] FIG8 is a schematic diagram of the real-time signal curve processing results according to Example 1 of the present invention; wherein, (A) the real-time signal curve processing results of well A01; (B) the real-time signal curve processing results of well A08; (C) the real-time signal curve processing results of well A12; (D) the TTP value data table of a 96-well PCR plate;

[0126] 9 is a schematic diagram summarizing the processing results of the real-time signal curve for each well of a 96-well plate according to Example 1 of the present invention;

[0127] Figure 10 is a schematic diagram of the real-time signal curve processing results in Example 2 of the present invention; wherein: (A) original signal curve; (B) step c, positive original signal curve; (C) step d, negative original signal curve; (D) step e, polynomial fitting average baseline graph; (E) step e, correction and initialization signal curve graph. (F) steps e and h, normalized signal curve spline fitting graph;

[0128] FIG11 is a schematic diagram of the real-time signal curve processing results according to Example 2 of the present invention; wherein, (A) the real-time signal curve processing results of well A01; (B) the real-time signal curve processing results of well A08; (C) the real-time signal curve processing results of well A12; (D) the TTP value data table of a 96-well PCR plate;

[0129] FIG12 is a schematic diagram summarizing the processing results of the real-time signal curve of each well of a 96-well plate according to Example 2 of the present invention. DETAILED DESCRIPTION

[0130] 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.

[0131] In this document, unless otherwise specified, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0132] 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.

[0133] Herein, the amplification container includes: a single PCR tube, an 8-well tube strip, a 96-well or 384-well PCR plate, a droplet, a microfluidic amplification pool, or other amplification containers suitable for PCR reactions.

[0134] In this article, the 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 tube row, a single well in a 96- or 384-well PCR plate, etc.

[0135] 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 needed), and other equivalent methods that can achieve the same effect are applicable.

[0136] In this article, the term "loop-mediated isothermal amplification (LAMP)" refers to 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.

[0137] Currently, the sequence-specific labeling methods used in loop-mediated amplification (LAMP) technology mainly include fluorescence of loop primer upon self-dequenching, HyBeacon probes, guanine quenching probes, 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 probes (OSD), molecular beacons, light cycler probes, assimilating probes, and mediator displacement probes. Different labeling methods are divided into two technical solutions: endpoint detection and real-time detection. Because these methods are based on different principles, technical solutions, operating procedures, and detection equipment and instruments, and lack universal technical standards, they cannot meet the requirements of standardization, automation, and high throughput in nucleic acid detection scenarios. Therefore, the present invention proposes a method suitable for classifying sparse signal amplification containers. It determines whether a specific amplification container is amplifying positive or negative. Based on this, the time to report positive amplification (TTP value) is determined based on the time point information.

[0138] In one example of the present application, an isothermal amplification reaction is performed in an isothermal amplification container to determine whether the container is positively amplified. If positive amplification is positive, the method for determining the positive reporting time is as follows:

[0139] Specifically, an isothermal amplification reaction is performed in a plurality of isothermal amplification containers; and for each of the plurality of isothermal amplification containers, signal acquisition is performed at a given time point to obtain an original real-time signal data set; based on the original real-time signal data set, the original real-time signal data is expanded to obtain an expanded set of original real-time signal data; based on the expanded set of original real-time signal data, at least one of the plurality of 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 classify the amplification container. Correcting at least a portion of the real-time signal data set to obtain a corrected real-time signal data set; performing secondary classification on at least one of the multiple isothermal amplification containers based on the corrected real-time signal data set to obtain a final judgment amplification container and a final judgment non-amplification container; and constructing a data function of the corrected real-time signal relative to the sampling time based on at least a portion of the multiple isothermal amplification containers; and 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 reporting time (TTP value) of a given amplification container corresponding to the signal threshold based on the data function.

[0140] It should be noted that the amplification baseline is determined from all negative data from the same channel. For example, in an 8 (rows) x 12 (columns) PCR plate, the first row is used for amplification testing of the FAM (fluorescent probe) channel, and the second row is used for amplification testing of the ROX (fluorescent probe) channel. The amplification baseline for the FAM channel is calculated from the negative data in the first row, and the amplification baseline for 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, the amplification baseline for that channel is calculated from all negative data in the PCR plate.

[0141] in,

[0142] For a given isothermal amplification container, the primary classification is performed by: determining a first signal threshold for the isothermal amplification based on a signal at at least one initial time point; and performing a primary classification of the isothermal amplification container between an amplification container and a non-amplification container based on a difference between the signal at other time points of the isothermal amplification container and the first signal threshold; for a given isothermal amplification container, the secondary classification is performed by the following steps: determining a second signal threshold based on a corrected signal at at least one initial time point; and performing a secondary classification of the isothermal amplification container between an amplification container and a non-amplification container based on a difference between the signal at other time points of the isothermal amplification container and the second signal threshold.

[0143] Specifically, for ease of understanding, the technical solution of the present application (taking real-time signal data analysis of self-circulating strand displacement amplification (LAMP) 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: Several amplification detection systems are prepared according to different sample types, such as negative quality control, positive quality control, and test samples, and packaged in specific amplification containers. The samples are then loaded onto a machine (a real-time fluorescence thermal cycler or real-time fluorescence constant temperature device, such as the Hangzhou Biori FQD-96A, Shanghai Hongshi SLAN-96S, or the Thermo Fisher Scientific ABI-7500) to initiate self-circulating chain displacement amplification. The detection device is run according to the preset real-time detection temperature control and signal acquisition program to obtain raw real-time signal data S (S contains A × T real-time signal data) from all amplifiers (amplifier refers to the smallest amplification unit in the amplification container, such as each single PCR tube, a single tube in an 8-well strip, a single well in a 96- or 384-well PCR plate, etc., with the total number of amplifiers being denoted as A) in each channel of the real-time detection instrument. The data matrix is ​​then saved in a storage medium, where T is set to be significantly smaller than the default or conventional value T0, but the duration of the amplification reaction remains unchanged. For example, for an amplification reaction running for 48 minutes, the conventional value T0 is set to 25, that is, the real-time signal is collected once every 2 minutes on average. In order to further shorten the running time of the nucleic acid amplification real-time detection technical solution, the total number of real-time signal readings T can be reduced to 12, that is, the real-time signal is collected once every 4 minutes on average (the running time of the device reading the signal can be shortened by half);

[0145] 2) Data expansion: The computer device reads the data matrix in the storage medium and obtains the original signal data of all amplifiers in a specific amplification container. According to the T real-time signal data (A1, A2, ..., A T ) calculate (using fitting, interpolation, regression or averaging methods) to obtain the continuous real-time signal curve data or expand it into (T+E) real-time signal data with higher data density (B1, B2, ..., B T+E ), 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 denoted as S';

[0146] 3) Initial classification judgment: select specific N data (B'1, B'2, ..., B' N, N is a preset value (e.g., N can be 5 or 6, etc.), calculate the signal threshold S'_thresh for each amplifier or all amplifiers, compare the subsequent real-time signal data of all N data with the signal threshold S'_thresh, and determine whether amplification has occurred in that amplifier (if the threshold is larger, it is determined that amplification has not occurred; if the threshold is smaller, it is determined that amplification has occurred). Perform the above steps on all amplifiers in sequence and complete the determination. A negative result is determined for amplification, and a positive result is determined for amplification.

[0147] 4) Correction and initial normalization: Based on the B' of the negative amplifier neg Set real-time signal data and get B' neg ×(T+E) 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 correction real-time signal data of all amplifiers is initialized (the baseline correction real-time signal data S' of each amplifier is initialized). flat According to the initial data point alignment) 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] 5) Secondary classification determination and positive time (TTP value) calculation: Use the method in step 3) 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 are compared with the signal threshold S' norm _thresh is used to compare the size and the amplification results of all amplifiers are judged again. The results of non-amplification are judged as negative results and those of amplification are judged as positive results. Determine the initial normalized real-time signal curve of each positive amplifier and the signal threshold S' norm The initial normalized real-time signal curve of each negative amplifier is compared with the signal threshold S'. norm _thresh does not have an intersection point, so the positive reporting time TTP value is set to infinity (Inf).

[0149] It should be noted that the above-described analytical method is not limited by different labeling methods, operating procedures, or detection equipment and instruments. Through calibration and initial normalization, the above-described method can automatically process and determine the real-time signal data from various amplifiers for different sample types but with the same or similar biochemical systems. It also calculates a universal quantifiable indicator, the time to positive signal, to characterize the onset of positive signals, thereby enabling semi-quantitative or quantitative analysis of test results.

[0150] The 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 limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0151] Example 1: Algorithm Verification Based on Single-Channel Loop-Mediated Isothermal Amplification Real-Time Signal (Data Analysis Process 1)

[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 the loop-mediated isothermal amplification (LAMP) experimental method to prepare the MGI (HuaDa Intelligent Manufacturing) self-developed duplex probe system for single-channel detection of nucleic acid templates containing several different concentrations. The total volume of the detection system is 30μl, of which 12μl of template is contained. The amplification container is a 96-well PCR plate, which contains 4 positive quality controls, 4 negative quality controls, and 80 simulated samples of 4 low-concentration nucleic acid templates. The concentrations of the preservative solution samples before extraction are 500, 400, 300, and 200 copies / ml, respectively, and each concentration contains 20 technical replicates. There are a total of 88 valid test wells, and the 11th column of the 96-well PCR plate is a vacant column. The purpose of the experiment is to test the detection performance of the amplification system after a certain formula substance is changed. The real-time signal acquisition device was a Hangzhou Biori FQD-96A, running the device client software Gene-9660. The acquisition process was divided into three phases: pre-acquisition, reverse transcription, and isothermal amplification. A total of 25 signal acquisition / reading time points were set, with readings occurring every 2 minutes, for a theoretical total run time of 48 minutes. The temperature was set to 30°C for signal acquisition and 65°C for reverse transcription and isothermal amplification. Click "Start Run" to initiate the acquisition process. Wait for a while until the acquisition process completes 25 acquisition steps and acquires all real-time amplification signal data. The client software Gene-9660 then automatically plots the real-time amplification signal curve shown in Figure 6. While the client software Gene-9660 does not analyze LAMP real-time signals, it can be used to export the raw LAMP real-time signal data to Excel or a text file and save it for analysis.

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

[0155] a. Read the LAMP real-time signal raw data from the Excel or text format file in step 1) and generate a two-dimensional matrix S of real-time signal raw data (the matrix contains 96 × 25 real-time signal data) based on the signal acquisition time point and the amplified real-time signal value;

[0156] b. Use the smoothing spline fitting method to fit the real-time signal raw data of all amplifiers to obtain the real-time signal smooth curve corresponding to each amplifier. Based on the real-time signal smooth curve data S, the expanded real-time signal fitting data S' is obtained. For example, the 96×25 two-dimensional matrix of real-time signal raw data is expanded to 96×2401, that is, the 25 sampling numbers between [1,25] (1, 2, 3, ..., 24, 25) are expanded to 2401 (1, 1.01, 1.02, ..., 24.99, 25);

[0157] c. Take the real-time signal fitting values ​​of the 1st, 101st, 201st, 301st, 401st and 501st signal sampling points of each amplifier in the fitting data S' 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 fitting values ​​of the above 6 signal acquisition time points of each amplifier are calculated;

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

[0160] d. Calculate the maximum value of the last 1900 real-time signal fitting values ​​(502nd to 2401st) for each amplifier, compare it with the corresponding signal threshold S'_thresh, and record the threshold comparison result (a value greater than the threshold is recorded as 1, and a value 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);

[0161] e. Calculate the average baseline of the unamplified real-time signal fitting data using a polynomial fit (with the polynomial order set to 8) for the corresponding real-time signal fitting values ​​of all negative amplifiers. Process the real-time signal fitting data two-dimensional matrix based on the aforementioned average baseline data to obtain a baseline-corrected real-time signal fitting data two-dimensional matrix (the matrix contains 96 × 2401 real-time signal data).

[0162] f. Initialize the baseline-corrected real-time signal fitting data two-dimensional matrix according to the real-time signal fitting value at the first signal acquisition time point (data alignment), and normalize all signal data values ​​in the initialized fitting data two-dimensional matrix to between [0, 1]. Obtain the initial normalized real-time signal data two-dimensional matrix;

[0163] g. Based on the two-dimensional matrix of initial normalized real-time signal data, take the initial normalized real-time signal values ​​of the 1st, 101st, 201st, 301st, 401st, and 501st signal sampling points of each amplifier to calculate the initial normalized signal threshold of each amplifier (a total of 96). The calculation steps are as follows:

[0164] First, calculate the average value (AVG) of the initial normalized real-time signal value of the above 6 signal sampling points of each amplifier 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'norm_thresh of each amplifier;

[0166] h. Calculate the maximum value of the last 1900 (502nd to 2401st) initial normalized real-time signal values ​​for each amplifier, compare it with the corresponding initial normalized signal threshold S'norm_thresh, and record the threshold comparison result (record it as 1 if it is greater than the threshold, and record it as 0 if it is less than the threshold). Based on the threshold comparison result, the amplifier with a value of 1 is preliminarily determined to be positive (amplified), and the amplifier with a value of 0 is preliminarily determined to be negative (not amplified);

[0167] i. Convert the initial normalized real-time signal data of each amplifier into an initial normalized real-time signal curve, 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 the real-time signal smooth curve of each negative amplifier does not have an intersection with the corresponding threshold, its positive reporting time (TTP value) is set to infinity (Inf);

[0168] j. Finally, the real-time signal curve processing results of each stage are shown in Figures 7 to 9. The TTP value data of the well position distribution of the 96-well plate obtained after processing and analyzing according to steps a to j are shown in Table 1.

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

[0170] Note: PC means positive; NC means negative.

[0171] Based on the above results, it can be calculated that the average TTP values ​​of the simulated samples with four low-concentration nucleic acid templates of 500, 400, 300, and 200 copies / ml, as well as the positive quality control and negative quality control groups are 8.95, 9.13, 9.55, 9.28, 7.24, and Inf, respectively. The corresponding coefficients of variation of the TTP values ​​within the group are 4.26%, 8.57%, 15.78%, 26.71%, 1.86%, and 0, respectively.

[0172] Example 2: Algorithm Verification Based on Single-Channel Loop-Mediated Isothermal Amplification Real-Time Signal (Data Analysis Process 2)

[0173] According to the results obtained in the embodiments of the present invention, considering that the total number of real-time signal reading or acquisition times is reduced, the running time of the nucleic acid amplification real-time detection technical solution can be further shortened (i.e., the total number of signal acquisition / reading time points of the acquisition program is reduced, for example, the original 25 signal acquisition / reading time points are reduced to 12, that is, the theoretical total running time is kept unchanged at 48 minutes, and the frequency of reading every 2 minutes is reduced to reading every 4 minutes), thereby reducing the time cost of the device in signal acquisition or reading.

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

[0175] To verify the feasibility of this method, this example, based on Example 1, deletes the odd-numbered signal data (1, 3, 5, ..., 23, 25, a total of 13 times) from the 96 × 25 real-time signal data read in step a of Example 1, retaining only the even-numbered signal data (2, 4, 6, ..., 22, 24, a total of 12 times), ultimately reducing the number of real-time signal data to 96 × 12. In subsequent steps, the updated real-time signal data will be used as the raw data for processing and analysis in this example, and compared with the calculation results of Example 1.

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

[0177] a. Read the LAMP real-time signal raw data from the Excel or text format file in step 1), and generate a two-dimensional matrix S of real-time signal raw data (the matrix contains 96×12 real-time signal data) based on the signal acquisition time point and the amplified real-time signal value;

[0178] b. Use a smoothing spline fitting method to fit the real-time signal raw data of all amplifiers to obtain the real-time signal smooth curve corresponding to each amplifier. Based on the real-time signal smooth curve data S, the expanded real-time signal fitting data S' is obtained. For example, a 96×12 two-dimensional matrix of real-time signal raw data is expanded to 96×2401, that is, the 12 sampling numbers between [1,12] (1, 2, 3, ..., 11, 12) are expanded to 2401 (1, 1.01, 1.02, ..., 24.99, 25);

[0179] c. Take the real-time signal fitting values ​​of the 1st, 101st, 201st, 301st, 401st and 501st signal sampling points of each amplifier in the fitting data S' 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 fitting values ​​of the above 6 signal acquisition time points of each amplifier are calculated;

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

[0182] d. Calculate the maximum value of the last 1900 real-time signal fitting values ​​(502nd to 2401st) for each amplifier, compare it with the corresponding signal threshold S'_thresh, and record the threshold comparison result (a value greater than the threshold is recorded as 1, and a value 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);

[0183] e. Calculate the average baseline of the unamplified real-time signal fitting data using a polynomial fit (with the polynomial order set to 8) for the corresponding real-time signal fitting values ​​of all negative amplifiers. Process the real-time signal fitting data two-dimensional matrix based on the aforementioned average baseline data to obtain a baseline-corrected real-time signal fitting data two-dimensional matrix (the matrix contains 96 × 2401 real-time signal data).

[0184] f. Initialize the baseline-corrected real-time signal fitting data two-dimensional matrix according to the real-time signal fitting value at the first signal acquisition time point (data alignment), and normalize all signal data values ​​in the initialized fitting data two-dimensional matrix to between [0, 1]. Obtain the initial normalized real-time signal data two-dimensional matrix;

[0185] g. Based on the two-dimensional matrix of initial normalized real-time signal data, take the initial normalized real-time signal values ​​of the 1st, 101st, 201st, 301st, 401st, and 501st signal sampling points of each amplifier to calculate the initial normalized signal threshold of each amplifier (a total of 96). The calculation steps are as follows:

[0186] First, calculate the average value (AVG) of the initial normalized real-time signal value of the above 6 signal sampling points of each amplifier 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'norm_thresh of each amplifier;

[0188] h. Calculate the maximum value of the last 1900 (502nd to 2401st) initial normalized real-time signal values ​​for each amplifier, compare it with the corresponding initial normalized signal threshold S'norm_thresh, and record the threshold comparison result (record it as 1 if it is greater than the threshold, and record it as 0 if it is less than the threshold). Based on the threshold comparison result, the amplifier with a value of 1 is preliminarily determined to be positive (amplified), and the amplifier with a value of 0 is preliminarily determined to be negative (not amplified);

[0189] i. Convert the initial normalized real-time signal data of each amplifier into an initial normalized real-time signal curve, 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 the real-time signal smooth curve of each negative amplifier does not have an intersection with the corresponding threshold, its positive reporting time (TTP value) is set to infinity (Inf);

[0190] j. Finally, the real-time signal curve processing results of each stage are shown in Figures 10 to 12. The TTP value data of the well position distribution of the 96-well plate obtained after processing and analyzing according to steps a to j are shown in Table 2.

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

[0192] Note: PC means positive; NC means negative.

[0193] Based on the above data results, the average TTP values ​​of the simulated samples of 4 low-concentration nucleic acid templates of 500, 400, 300 and 200 copies / ml, as well as the positive quality control and negative quality control groups were calculated to be 9.15, 9.37, 9.53, 9.14, 7.29 and Inf, respectively. The corresponding coefficients of variation of the TTP values ​​within the group were 5.43%, 8.34%, 14.88%, 25.74%, 4.66% and 0, respectively.

[0194] In summary, by comparing the data analysis results of Example 1 and Example 2, it can be concluded that the calculation results of data analysis process one and data analysis process two are basically consistent, indicating that the optimized data analysis process is more suitable for the judgment of sparse real-time signal results, and can achieve a lower number or frequency of real-time signal reading / collection, further shortening the running time of the nucleic acid amplification real-time detection technology solution.

[0195] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0196] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative 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 sparse signal amplification containers, characterized in that: include: For each of the multiple amplification containers, respectively, signal acquisition is performed at a given time point 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 amplification 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 Classifying the amplification container between an amplification container and a non-amplification container according to a first difference between the signal of the amplification container at other time points and the first signal threshold; 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 Based on a second difference between the signal of the amplification container at another time point and the second signal threshold, the amplification container is secondarily classified into an amplification container and a non-amplification container.

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 Wherein, AVG represents the average value of the amplified real-time signal data selected for a classification; STD represents the standard deviation of the amplified real-time signal data selected for a classification; and 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.

5. The method according to claim 4, characterized in that 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 the other time point is less than the first signal threshold, which is an indication that the amplification container is initially judged to be a non-amplification container.

6. 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.

7. The method according to claim 2 or 6, 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.

8. The method according to claim 7, characterized in that 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 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.

9. The method according to claim 1, characterized in that: Before the first classification, the method further includes performing expansion processing on the original real-time signal data set.

10. The method according to claim 9, characterized in that The expansion process is achieved by performing data processing on the data matrix of the original real-time signal data set; The data processing is performed using at least one of fitting, interpolation, regression or averaging methods.

11. 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; and According to the method according to any one of claims 1 to 10, the plurality of amplification containers are classified to determine final judgment amplification containers and final judgment non-amplification containers; and 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; and 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, the positive reporting time of a given amplification container corresponding to the signal threshold is determined.

12. 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 11, 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.

13. 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 11, 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.

14. The method according to any one of claims 1 or 11 to 13, characterized in that: The amplification is selected from isothermal amplification; the isothermal amplification is selected from at least one of loop-mediated amplification, rolling amplification, strand displacement amplification, multiple displacement amplification, recombinase polymerase amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.

15. A device suitable for sparse signal amplification container classification, characterized in that: include: A data collection unit, 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 data expansion unit, the data expansion unit is connected to the data collection unit and performs expansion processing on the original data to obtain an amplified real-time signal data set; A first classification determination unit, wherein 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 amplification real-time signal data set, so as to obtain a preliminarily determined amplification container and a preliminarily determined non-amplification container; A correction unit, the correction module is connected to the first classification and determination module, and is used to 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 amplification real-time signal data set, so as to obtain a corrected real-time signal data set; The second classification determination unit, wherein the second classification determination module is connected to the correction module, 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.

16. 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 15, connected to the plurality of amplification containers, for sorting the amplification containers; and a data function construction device connected to the device of claim 15 and configured to construct 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.

17. 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 16, 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.

18. 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 16 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.

19. 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 14 is executed.

20. 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 14.

21. 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 14.

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