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WO2025091342A1 - Procédé et dispositif appropriés pour la classification de conteneurs d'amplification de signal épars - Google Patents

Procédé et dispositif appropriés pour la classification de conteneurs d'amplification de signal épars Download PDF

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Publication number
WO2025091342A1
WO2025091342A1 PCT/CN2023/129149 CN2023129149W WO2025091342A1 WO 2025091342 A1 WO2025091342 A1 WO 2025091342A1 CN 2023129149 W CN2023129149 W CN 2023129149W WO 2025091342 A1 WO2025091342 A1 WO 2025091342A1
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Prior art keywords
amplification
signal
time
container
real
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Chinese (zh)
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夏贇
耿春雨
李景
刘健
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to PCT/CN2023/129149 priority Critical patent/WO2025091342A1/fr
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis

Definitions

  • the present invention relates to the field of bioinformatics analysis and molecular biology detection, and in particular, to a method and a device suitable for classifying sparse signal amplification containers.
  • PCR Polymerase chain reaction
  • PCR is the most commonly used method for rapid and large-scale replication of target nucleic acid molecules in vitro or in a test tube. It can be used to amplify specific DNA fragments and realize qualitative and quantitative detection of biochemical analytes such as nucleic acids.
  • PCR uses variable temperature cycles to allow the double-stranded DNA 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, which can theoretically 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 the level that the instrument sensor can detect.
  • nucleic acid isothermal amplification has become an important diagnostic tool, not only for clinical applications, but also for food quality control and environmental monitoring.
  • Nucleic acid isothermal amplification technology is a new type of solution proposed to overcome the various limitations and shortcomings in the application of PCR technology. They can quickly expand the number of copies of target DNA or RNA fragments at a constant temperature. This type of technology can reduce the complicated steps of sample and reagent preparation, completely get rid of the dependence on thermal cyclers, and can greatly simplify the complexity of nucleic acid detection schemes, which is of great significance for on-site instant detection applications.
  • LAMP loop-mediated amplification
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • MDA multiple displacement amplification
  • RPA recombinase polymerase amplification
  • TMA transcription mediated amplification
  • SPIA single primer isothermal amplification
  • HDA helicase dependent amplification
  • the polymerase-mediated nucleic acid chain displacement reaction is a technology widely used in nucleic acid amplification reactions, and it is also a simulation and variant reaction of the nucleic acid replication process in organisms.
  • the process mainly includes two processes: primer extension under the action of a polymerase with chain displacement activity and the replacement of the original nucleic acid chain downstream by the new nucleic acid chain generated by the extension.
  • the polymerase-mediated chain displacement leads to the unwinding of the original double-stranded chain and the generation of a new double-stranded chain, resulting in an amplification reaction.
  • This displacement reaction replaces the high-temperature unwinding process in the traditional variable temperature reaction, can be carried out in a wide temperature range, and is simple and convenient to operate.
  • the nucleic acid chain displacement reaction has been widely used in various fields of molecular biology due to its high specificity and high sensitivity detection characteristics, and has also received great attention in the amplification of detection signals and diagnostic biosensor detection.
  • Self-circulating chain displacement amplification includes LAMP, RCA, MDA, SDA, SPIA and other technologies. Among them, the most widely used LAMP technology has the characteristics and advantages of typical self-circulating chain displacement amplification, and the amplification efficiency and sensitivity are also the best.
  • the real-time detection temperature control and signal acquisition procedures adopted by each technical solution based on different labeling methods are different, resulting in differences in their respective acquisition signals, and the real-time detection results cannot be compared in parallel.
  • the real-time detection procedures of each technical solution with the same labeling method are artificially preset, and the preset number of readings and reading time intervals are relatively arbitrary, and there is a lack of unified standards, which leads to the total detection time being arbitrary and usually long.
  • 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.
  • the commonly used strategy is to reduce the number or frequency of real-time signal reading or collection as much as possible (in the extreme case, only one signal reading/collection is performed before and after the amplification reaction is started, that is, it is degraded to the endpoint detection technical solution).
  • the reduction in the number or frequency of real-time signal reading/collection will simultaneously bring about the loss of amplification reaction kinetic information, and the subtle changes in real-time signals cannot be accurately portrayed.
  • 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.
  • the present invention proposes a method suitable for sparse signal amplification container classification.
  • the method includes: for each of a plurality of amplification containers, signal acquisition is performed at a given time point respectively, so as to obtain an original real-time signal data set; based on the original real-time signal data set, a classification is performed for at least one of the plurality of amplification containers, so as to obtain a preliminary judgment amplification container and a preliminary judgment non-amplification container; based on at least a portion of the signal of the preliminary judgment non-amplification container, an amplification baseline is determined, and at least a portion of the amplification real-time signal data set is corrected using the amplification baseline, so as to obtain a corrected real-time signal data set; based on the corrected real-time signal data set, a secondary classification is performed for at least one of the plurality of amplification containers, so as to obtain a final judgment amplification container and a final judgment non-a
  • the method simplifies the existing processing method of amplification real-time detection signals, and uses existing biochemical experimental data to optimize and iterate algorithms, improves calculation accuracy, data processing efficiency and versatility of analysis methods, is more suitable for the result determination analysis of sparse real-time signals, and realizes a rapid and automated analysis method for determining nucleic acid detection results in various amplification containers (such as 96-well PCR plates), and forms a technical standard.
  • the amplification baseline can be obtained by calculation methods such as fitting, interpolation, regression or averaging.
  • the given time points are not less than 3.
  • the given number of time points is selected from the minimum number of time points that does not affect the analysis result.
  • 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.
  • the calculation of the continuous real-time signal curve data corresponding to each amplification container is to obtain a real-time fluorescence intensity curve by continuously monitoring the fluorescence signal during the amplification reaction.
  • key amplification feature points (such as starting point, exponential growth section, platform section, etc.) can be identified, so as to calculate the quality parameters of the amplification reaction (such as positive reporting time, amplification efficiency, etc.).
  • the above method for classifying sparse signal isothermal amplification containers may also include at least one of the following technical features:
  • the primary classification is performed by: determining the first signal threshold of the amplification based on the 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 the first difference between the signal at other time points of the amplification container and the first signal threshold; for a given amplification container, the secondary classification is performed by: determining the second signal threshold based on the 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 the second difference between the signal at other time points of the amplification container and the second signal threshold.
  • the method of using the two classifications can analyze the 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.
  • the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.
  • the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool.
  • any container that can achieve nucleic acid amplification can be used as an amplification container.
  • 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
  • N1 is an integer.
  • N1 includes integers from 1 to 25. Specifically, N1 includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N1 is set to 10.
  • the above signal threshold calculation method is only exemplary.
  • the signal threshold can also be calculated by variance or coefficient of variation.
  • the first difference is determined by comparing the signal of the amplification container at other time points with the first signal threshold.
  • the signal at other time points is greater than the first signal threshold, which is an indication that the amplification container is a preliminarily judged amplification container; the signal at other time points is less than the first signal threshold, which is an indication that the amplification container is a preliminarily judged non-amplification container.
  • the initial judgment of amplification in the present application is obtained through comprehensive judgment, that is, as long as the signal result at one time point is greater than the first signal threshold, it is considered to be initial judgment of positive amplification; similarly, the initial judgment of non-amplification (initial judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results at all time points are less than the first signal threshold.
  • AVG norm represents the average value of the corrected real-time signal data selected for secondary classification
  • STD norm represents the standard deviation of the corrected real-time signal data selected for secondary classification
  • N 2 is an integer.
  • N2 includes integers of 1 to 25. Specifically, N2 includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N2 is 10.
  • the above signal threshold calculation method is only exemplary.
  • the signal threshold can also be calculated by variance or coefficient of variation.
  • the values of N1 and N2 may be the same or different, and are generally set based on actual experimental requirements.
  • the second difference is determined by comparing the signal of the amplification container at other time points with the second signal threshold.
  • the signal at other time points is greater than the second signal threshold, which is an indication that the amplification container is a final-judgment amplification container; the signal at other time points is less than the second signal threshold, which is an indication that the amplification container is a final-judgment non-amplification container.
  • the final judgment of amplification (final judgment of positive amplification) described in the present application is obtained through comprehensive judgment, that is, the signal result of any one of the other time points is greater than the second signal threshold, which is considered to be the final judgment of positive amplification; similarly, the final judgment of non-amplification (final judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results of other time points are all less than the second signal threshold.
  • 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.
  • the method before the primary classification, 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, that is, more data points are obtained at a specific time point.
  • the increase in data density is beneficial to providing more accurate and detailed signal information, making subsequent classification, analysis and prediction more accurate.
  • the expansion processing is realized by performing data processing on the data matrix of the original real-time signal data set; the data processing is performed by using at least one of fitting, interpolation, regression or averaging methods. Performing data expansion processing by fitting, interpolation, regression or averaging methods can fill the gaps in signal data, making the real-time signal data more continuous and substantial.
  • the data matrix of the original real-time signal data set is read to obtain T real-time signal data of the amplifier. Then, any calculation method such as fitting, interpolation, regression or averaging is used to expand the obtained T real-time signal data into (such as T+E) real-time signal data with higher data density, where E is a positive integer.
  • the present invention proposes a method for determining the time (TTP value) of amplification reaction reporting positive.
  • the method includes: performing an amplification reaction in a plurality of amplification containers; and the method described in the first aspect of the present application or any embodiment, classifying the plurality of amplification containers, 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 positive time of a given amplification container corresponding to the signal threshold based on the data function.
  • the method improves the calculation accuracy and experimental efficiency of the isothermal amplification reaction reporting positive time (TTP value) through an automated and efficient data processing scheme
  • 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.
  • the method for determining the positive time of an amplification reaction may further include at least one of the following technical features:
  • the data function can be calculated and obtained by polynomial fitting, spline fitting, exponential fitting or other equivalent methods.
  • the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.
  • the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool.
  • any container that can achieve nucleic acid amplification can be used as an amplification container.
  • 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.
  • the present invention proposes a method for analyzing biological samples by amplification reaction.
  • 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.
  • the method has the advantages of high sensitivity, high efficiency and rapidity, low cost, easy automation and wide adaptability, making it a very valuable analytical tool in the fields of biological research, medical diagnosis and environmental monitoring.
  • the method can also be applied to the analysis of different biological samples (such as the expression level of the target gene, the efficiency of the kit or primer, the sample purity and dilution degree or the comparison between samples, etc.), and has the advantages of short analysis time and high sensitivity.
  • the above method for analyzing a biological sample by an amplification reaction may further include at least one of the following technical features:
  • the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.
  • the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool.
  • any container that can achieve nucleic acid amplification can be used as an amplification container.
  • 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.
  • the present invention proposes a method for determining the content of nucleic acid in a nucleic acid sample.
  • 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 content of nucleic acid in the nucleic acid sample based on the positive reporting time.
  • 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 content of nucleic acid in a nucleic acid sample.
  • the method can determine the content of nucleic acid in the test nucleic acid sample based on the positive reporting time, and the method has a wide applicability and can be used to parallel compare the test results obtained based on different labeling methods; it is also suitable for analyzing the limitations of result judgment caused by differences in detection procedures in the same labeling method.
  • the method for determining the nucleic acid content in a nucleic acid sample may further include at least one of the following technical features:
  • the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.
  • the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool.
  • any container that can achieve nucleic acid amplification can be used as an amplification container.
  • the amplification is selected from isothermal amplification.
  • the isothermal amplification is selected from loop-mediated amplification, rolling amplification, strand displacement amplification, multiple displacement amplification, At least one of polymerase amplification, transcription-mediated amplification, single primer isothermal amplification and helicase-dependent amplification.
  • the present invention provides a method for determining the content of a target nucleic acid molecule in a nucleic acid sample.
  • the method comprises: using primers for the target nucleic acid molecule, performing an isothermal amplification reaction in multiple isothermal amplification containers; determining the positive reporting time of a given amplification container according to the method described in the second aspect; and determining the content of the target nucleic acid molecule in the nucleic acid sample based on the positive reporting time.
  • the method is suitable for low-quality and low-concentration nucleic acid samples, and does not require professional instruments and complicated 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 the detection; and the content of the target nucleic acid molecule is determined based on the positive reporting time, avoiding the need for accurate and quantitative thermal cycling amplification reactions in traditional PCR methods, thereby reducing the difficulty and time cost of experimental operations.
  • the method for determining the content of target nucleic acid molecules in a nucleic acid sample may further include at least one of the following technical features:
  • the method can quickly calculate the expression level of the target gene in the test nucleic acid sample based on the positive reporting time.
  • the amplification container is selected from at least one of a single PCR tube, an 8-well tube, a 96-well or 384-well PCR plate, a droplet, and a microfluidic amplification pool.
  • the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool.
  • any container that can achieve nucleic acid amplification can be used as an amplification container.
  • 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.
  • the present invention proposes a device suitable for sparse signal amplification container classification.
  • the device includes: a data collection unit, which is used to collect signals at multiple given time points for each of a plurality of amplification containers, so as to obtain an original real-time signal data set; a data expansion unit, which is connected to the data collection unit and performs expansion processing on the original data, so as to obtain an amplification real-time signal data set; a first classification determination unit, the first classification predetermined module is connected to the data collection module, and is used to perform a classification for at least one of the plurality of amplification containers based on the amplification real-time signal data set, so as to obtain a preliminary judgment amplification container and a preliminary judgment non-amplification container; a correction unit, the correction module is connected to the first classification determination module, and is used to determine an amplification baseline based on at least a part of the signal of the preliminary judgment non-amplification container, and use the
  • the device can automatically collect the time point signal data of the sample to be tested in the amplification container without manual intervention, which can improve the experimental efficiency. Moreover, when the real-time signal obtained by the sample is small, accurate analysis can still be performed. In addition, the two classifications determine positive amplification and negative amplification, which improves the accuracy of classification. Since the above-mentioned device is based on machine learning technology, it can also perform self-learning and optimization to adapt to the amplification container classification tasks under different conditions.
  • the data collection unit S001 is connected to the data expansion unit S002, the data expansion unit S002 is connected to the first classification determination unit S003, the first classification determination unit S003 is connected to the correction unit S004, and the correction unit S004 is connected to the second classification determination unit S005.
  • the above-mentioned device suitable for sparse signal amplification container classification may also include at least one of the following technical features:
  • the primary classification is performed by: determining the first signal threshold of the amplification based on the 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 the first difference between the signal at other time points of the amplification container and the first signal threshold; for a given amplification container, the secondary classification is performed by: determining the second signal threshold based on the 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 the second difference between the signal at other time points of the amplification container and the second signal threshold.
  • the method of using the two classifications can analyze the 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.
  • the amplification container is selected from at least one of a single PCR tube, an 8-well tube row, a 96- or 384-well PCR plate, a droplet, and a microfluidic amplification pool.
  • the amplification container is not limited to the aforementioned single PCR tube, 8-row tube, 96 or 384-well PCR plate, droplet and microfluidic amplification pool.
  • any container that can achieve nucleic acid amplification can be used as an amplification container.
  • 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
  • N1 is an integer.
  • N1 includes integers from 1 to 25. Specifically, N1 includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N1 is 10.
  • the above signal threshold calculation method is only exemplary.
  • the signal threshold can also be calculated by variance or coefficient of variation.
  • the signal at other time points is greater than the first signal threshold, which is an indication that the amplification container is a preliminarily judged amplification container; the signal at other time points is less than the first signal threshold, which is an indication that the amplification container is a preliminarily judged non-amplification container.
  • the initial judgment of amplification in the present application is obtained through comprehensive judgment, that is, as long as the signal result at one time point is greater than the first signal threshold, it is considered to be initial judgment of positive amplification; similarly, the initial judgment of non-amplification (initial judgment of negative amplification) is also obtained through comprehensive judgment, that is, the signal results at all time points are less than the first signal threshold.
  • AVG norm represents the average value of the corrected real-time signal data selected for secondary classification
  • STD norm represents the standard deviation of the corrected real-time signal data selected for secondary classification
  • N 2 is an integer.
  • N2 includes integers of 1 to 25. Specifically, N2 includes: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some preferred embodiments of the present application, N2 is 10.
  • the values of N1 and N2 may be the same or different, and are generally set based on actual experimental requirements.
  • the signal at other time points is greater than the second signal threshold, which is an indication that the amplification container is a final-judgment amplification container; the signal at other time points is less than the second signal threshold, which is an indication that the amplification container is a final-judgment non-amplification container.
  • the present invention proposes a system for determining the time of reporting positive amplification reaction.
  • 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, and is used to classify the amplification containers; and a data function construction device, the data function construction device is connected to the device described in the sixth aspect of the present invention, and is used to construct a data function of the corrected real-time signal relative to the sampling time based on at least a part of the multiple amplification containers; and a signal threshold determination device for a final judgment non-amplification container, the signal threshold determination device for the 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 reporting positive time acquisition device, the
  • the system for determining the positive reporting time of an amplification reaction can accurately judge positive samples and negative samples, and can evaluate parameters such as sensitivity, specificity, precision, repeatability, reproducibility, and detection limit based on the obtained positive reporting time data, so that independent test results across amplification systems, instrument platforms, and laboratories have intra-batch and inter-batch result data comparability.
  • the present invention proposes a system for analyzing biological samples through amplification reactions.
  • the system includes: a biological sample acquisition device, the biological sample acquisition device is used to acquire a biological sample suspected of containing nucleic acid; and the system for determining the positive reporting time of an amplification reaction as described in the seventh aspect of the present invention, the system is connected to the biological sample acquisition device, and is used to determine the positive reporting time of a given amplification container; and an analysis device, the analysis device is connected to the system for determining the positive reporting time of an amplification reaction, and is used to analyze the nucleic acid content in the biological sample.
  • the advantage of the system for analyzing biological samples through amplification reactions is that it avoids the complicated data analysis process in traditional PCR technology, and the model structure can analyze biological samples quickly, accurately and automatically.
  • the present invention provides a system for determining the nucleic acid content in a nucleic acid sample.
  • the system comprises: the system for determining the positive reporting time of an amplification reaction as described in the seventh aspect of the present invention, which is used to determine the positive reporting time of a given amplification container; and a nucleic acid content acquisition device, which is connected to the system and is used to determine the nucleic acid content in the nucleic acid sample.
  • the system can determine the content of nucleic acid in a nucleic acid sample by the positive reporting time of the amplification reaction, thereby avoiding the tedious data analysis process and saving time and cost.
  • the present invention proposes a system for determining the content of target nucleic acid molecules in a nucleic acid sample.
  • the system comprises: the system for determining the positive reporting time of an amplification reaction as described in the seventh aspect of the present invention, which is used to determine the positive reporting time of a given amplification container; and a target nucleic acid molecular weight acquisition device, which is connected to the system and is used to determine the content of the target nucleic acid molecule in the nucleic acid sample.
  • the system X01 for determining the positive reporting time of the amplification reaction (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) is connected to the target nucleic acid molecular weight acquisition device S900.
  • the present invention provides a computer program product.
  • the computer program product includes computer instructions, and when part or all of the computer instructions are run on a computer, the method described in the first aspect, the second aspect, the third aspect, the fourth aspect, or the fifth aspect of the present invention is executed.
  • the computer program product includes analysis application software or a program compression package.
  • the present invention provides a computing device.
  • the computing device comprises: a memory and a processor; the memory is used to store a computer program; the processor is used to execute the computer program to implement the method described in the first aspect, the second aspect, the third aspect, the fourth aspect, or the fifth aspect of the present invention.
  • These various embodiments may include: being implemented in one or more computer programs, which may be executed and/or interpreted on a programmable system including at least one programmable processor, which may be a dedicated or general programmable processor, which may receive data and instructions from a storage system, at least one input device, and at least one output device, and transmit data and instructions to the storage system, the at least one input device, and the at least one output device.
  • a programmable processor which may be a dedicated or general programmable processor, which may receive data and instructions from a storage system, at least one input device, and at least one output device, and transmit data and instructions to the storage system, the at least one input device, and the at least one output device.
  • the program code for implementing the method disclosed in the present application can be written in any combination of one or more programming languages. These program codes can be provided to a processor or controller of a general-purpose computer, a special-purpose computer, or other programmable data processing device, so that the program code, when executed by the processor or controller, enables the functions/operations specified in the flow chart and/or block diagram to be implemented.
  • the program code can be executed entirely on the machine, partially on the machine, partially on the machine as a stand-alone software package and partially on a remote machine, or entirely on a remote machine or server.
  • 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, apparatus, or device.
  • 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, apparatus, or device, or any suitable combination of the foregoing.
  • machine-readable storage media would 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.
  • 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.
  • a display device e.g., a CRT (Cathode-Ray Tube) or LCD (Liquid Crystal Display) monitor
  • a keyboard and pointing device e.g., a mouse or trackball
  • 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).
  • the systems and techniques described herein may be implemented in a computing system that includes backend components (e.g., as a data server), or a computing system that includes middleware components (e.g., an application server), or a computing system that includes frontend components (e.g., a user computer with a graphical user interface or a web browser through which a user can interact with implementations of the systems and techniques described herein), or a computing system that includes any combination of such backend components, middleware components, or frontend components.
  • the components of the system may be interconnected by any form or medium of digital data communication (e.g., a communication network). Examples of communication networks include: LAN (Local Area Network), WAN (Wide Area Network), the Internet, and blockchain networks.
  • a computer system may include a client and a server.
  • the client and the server are generally remote from each other and usually interact through a communication network.
  • the relationship between the client and the server is generated by computer programs running on the corresponding computers and having a client-server relationship with each other.
  • the server may be a cloud server, also known as a cloud computing server or a cloud host, which is a host product in the cloud computing service system to solve the defects of difficult management and weak business scalability in traditional physical hosts and VPS services ("Virtual Private Server", or "VPS" for short).
  • the server may also be a server for a distributed system, or a server combined with a blockchain.
  • FIG1 is a schematic diagram of a device structure suitable for sparse signal amplification container classification according to one embodiment of the present invention
  • FIG. 2 is a schematic diagram of a system structure for determining the positive reporting time of an amplification reaction according to an embodiment of the present invention
  • FIG3 is a schematic diagram of a system structure for analyzing biological samples through an amplification reaction according to an embodiment of the present invention
  • 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.
  • FIG5 is a schematic diagram of the structure of a system for determining the content of target nucleic acid molecules in a nucleic acid sample according to one embodiment of the present invention
  • FIG6 is a single-channel LAMP real-time signal curve according to Example 1 of the present invention (Bori FQD-96A original data);
  • 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;
  • Example 8 is a schematic diagram of the real-time signal curve processing results in Example 1 of the present invention; wherein, (A) the real-time signal curve processing results of well position A01; (B) the real-time signal curve processing results of well position A08; (C) the real-time signal curve processing results of well position A12; (D) the TTP value data table of a 96-well PCR plate;
  • FIG. 9 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 1 of the present invention.
  • 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;
  • Example 11 is a schematic diagram of the real-time signal curve processing results in Example 2 of the present invention; wherein, (A) the real-time signal curve processing results of well position A01; (B) the real-time signal curve processing results of well position A08; (C) the real-time signal curve processing results of well position A12; (D) the TTP value data table of a 96-well PCR plate;
  • Example 12 is a schematic diagram summarizing the real-time signal curve processing results for each well of a 96-well plate in Example 2 of the present invention.
  • 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.
  • the amplification container includes: a single PCR tube, 8-well tubes, a 96-well or 384-well PCR plate, a droplet, a microfluidic amplification pool or other amplification containers suitable for PCR reactions.
  • LAMP loop-mediated isothermal amplification
  • this embodiment adopts a loop-mediated isothermal amplification (LAMP) experimental method, and prepares a single-channel detection of nucleic acid templates containing several different concentrations of MGI (MGI) self-developed double-stranded probe system.
  • the total volume of the detection system is 30 ⁇ l, which contains 12 ⁇ l of template.
  • 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.
  • step 1) 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 the real-time signal raw data according to the two dimensions of signal acquisition time point and amplified real-time signal value (the matrix contains 96 ⁇ 25 real-time signal data);
  • the corresponding real-time signal fitting values of all negative amplifiers are fitted with a polynomial (the polynomial order is set to 8) to calculate the average baseline of the unamplified real-time signal fitting data.
  • the two-dimensional matrix of the real-time signal fitting data is processed to obtain the two-dimensional matrix of the real-time signal fitting data after baseline correction (the matrix contains 96 ⁇ 2401 real-time signal data);
  • TTP value the positive reporting time of the amplifier. Since there is no intersection between the real-time signal smooth curve of each negative amplifier and the corresponding threshold, therefore, set its positive reporting time (TTP value) to infinity (Inf);
  • Table 1 96-well plate well position distribution TTP value data table
  • the average TTP values of the simulated samples with 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 are 8.95, 9.13, 9.55, 9.28, 7.24 and Inf, respectively, and the corresponding coefficients of variation of TTP values within the group are 4.26%, 8.57%, 15.78%, 26.71%, 1.86% and 0, respectively.
  • Example 2 Algorithm verification based on single-channel loop-mediated isothermal amplification real-time signal (data analysis process 2)
  • the running time of the nucleic acid amplification real-time detection technical solution can be further shortened (that is, the total number of signal collection/reading time points in the collection program is reduced, for example, the original 25 signal collection/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 collection or reading.
  • this embodiment deletes the odd-numbered signal data (1, 3, 5, ..., 23, 25, a total of 13 times) of the 96 ⁇ 25 real-time signal data read in step a of Example 1, and only retains the even-numbered signal data (2, 4, 6, ..., 22, 24, a total of 12 times), and finally reduces to 96 ⁇ 12 real-time signal data.
  • the subsequent steps will process and analyze the updated real-time signal data as the original data of this embodiment, and compare it with the calculation results of Example 1.
  • step 1) 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 the real-time signal raw data according to the two dimensions of signal acquisition time point and amplified real-time signal value (the matrix contains 96 ⁇ 12 real-time signal data);
  • the corresponding real-time signal fitting values of all negative amplifiers are fitted with a polynomial (the polynomial order is set to 8) to calculate the average baseline of the unamplified real-time signal fitting data.
  • the two-dimensional matrix of the real-time signal fitting data is processed to obtain the two-dimensional matrix of the real-time signal fitting data after baseline correction (the matrix contains 96 ⁇ 2401 real-time signal data);
  • TTP value the positive reporting time of the amplifier. Since there is no intersection between the real-time signal smooth curve of each negative amplifier and the corresponding threshold, therefore, set its positive reporting time (TTP value) to infinity (Inf);
  • Table 2 96-well plate well position distribution TTP value data table
  • 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 are 9.15, 9.37, 9.53, 9.14, 7.29 and Inf, respectively, and the corresponding coefficients of variation of TTP values within the group are 5.43%, 8.34%, 14.88%, 25.74%, 4.66% and 0, respectively.
  • Example 2 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 technical solution.

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Abstract

La présente invention concerne un procédé et un dispositif appropriés pour la classification de conteneurs d'amplification de signal épars. Le procédé consiste à : pour chacun d'une pluralité de conteneurs d'amplification, réaliser une acquisition de signal à un instant donné pour obtenir un ensemble de données de signal en temps réel d'origine ; sur la base de l'ensemble de données de signal en temps réel d'origine, réaliser une classification primaire sur au moins l'un de la pluralité de conteneurs d'amplification pour obtenir un conteneur d'amplification initialement déterminé et un conteneur de non-amplification initialement déterminé ; déterminer une ligne de base d'amplification sur la base d'au moins quelqu'uns des signaux du conteneur de non-amplification initialement déterminé, et utiliser la ligne de base d'amplification pour corriger au moins une partie de l'ensemble de données de signal en temps réel d'origine pour obtenir un ensemble de données de signal en temps réel corrigé ; et sur la base de l'ensemble de données de signal en temps réel corrigé, réaliser une classification secondaire sur au moins l'un de la pluralité de conteneurs d'amplification pour obtenir un conteneur d'amplification finalement déterminé et un conteneur de non-amplification finalement déterminé.
PCT/CN2023/129149 2023-11-01 2023-11-01 Procédé et dispositif appropriés pour la classification de conteneurs d'amplification de signal épars Pending WO2025091342A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101688250A (zh) * 2007-12-26 2010-03-31 爱科来株式会社 核酸扩增判定方法以及核酸扩增判定装置
WO2016052991A1 (fr) * 2014-10-01 2016-04-07 Seegene, Inc. Procédés d'analyse d'échantillons
US20170046480A1 (en) * 2015-08-14 2017-02-16 Tetracore, Inc. Device and method for detecting the presence or absence of nucleic acid amplification
CN113971068A (zh) * 2020-07-24 2022-01-25 中移(苏州)软件技术有限公司 资源调整方法、装置、设备和存储介质

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101688250A (zh) * 2007-12-26 2010-03-31 爱科来株式会社 核酸扩增判定方法以及核酸扩增判定装置
WO2016052991A1 (fr) * 2014-10-01 2016-04-07 Seegene, Inc. Procédés d'analyse d'échantillons
US20170046480A1 (en) * 2015-08-14 2017-02-16 Tetracore, Inc. Device and method for detecting the presence or absence of nucleic acid amplification
CN113971068A (zh) * 2020-07-24 2022-01-25 中移(苏州)软件技术有限公司 资源调整方法、装置、设备和存储介质

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