A digital pcr concentration calculation method
A calculation method and concentration technology, which are applied in the field of digital PCR concentration calculation, can solve the problem that the measurement accuracy cannot be guaranteed, and achieve the effect of broadening the concentration detection range and improving the accuracy.
Active Publication Date: 2020-08-21
领航医学科技(深圳)有限公司
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Problems solved by technology
[0003] Quantitative measurement of gene concentration by digital PCR based on Poisson distribution can have very high accuracy, but the measurement accuracy cannot be guaranteed for the dilution in the dynamic range of unknown samples
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[0019] Based on the real-time clustering concentration calculation method, multi-reaction point detection is adopted to monitor the reaction points in real time, and data detection is performed in each reaction cycle. The data expression of the detection reaction point can be quantifiable physical or chemical quantities such as light intensity, number of molecules, number of nucleic acids, number of proteins, etc. that have expression molecules or the number of single nucleic acids or proteins. This detection is a dynamic detection, from the beginning of the reaction to the end of the reaction, the reaction point data detection is performed. The multi-period detection data of each reaction point must be stored correspondingly, and then the amplification curve of the reaction point is drawn after the reaction amplification is completed. The amplification curve Figure one Generally, fluorescence amplification curve is the main method.
[0020] The initial target gene number diffe...
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The invention discloses a digital PCR concentration calculation method, which determines and calculates the initial concentration of a reactant according to the amplification cycleCt value of the real-time fluorescence quantitative curve in the PCR amplification process combined with the real-time clustering method. The fluorescence threshold R11 has an intersection with the amplification curve ofeach positive reaction unit during exponential growth, and the intersection corresponds to the corresponding amplification cycle value Cti. The digital PCR concentration calculation method specifically includes the following steps: clustering according to the Cti, obtaining k clusters, wherein the central value of each cluster is M1, M2, ..., Mk in descending order; calculating that the number ofthe Cti contained in each cluster is Sj, calculating the number of target genes in each cluster by an Mj and the Sj, adding up the number of target genes in each cluster, finally obtaining the concentration value of the initial target genes.
Description
Technical field [0001] The invention relates to a digital PCR concentration calculation method. Background technique [0002] Digital PCR is to evenly distribute the fluorescence quantitative reaction system to a large number of tiny reaction units, each of which does not contain or contains one or more target gene fragments. After the amplification, the positive detection signal is generated for the fragments containing the target gene, and the detection signal is not generated for the fragments without the target gene. The ratio of the number of positive reaction units judged by the end-point fluorescence signal to the total reaction units is calculated by statistical methods The number of copies of the target gene in the original sample. [0003] Digital PCR based on Poisson distribution can have very high accuracy for quantitative measurement of gene concentration, and it cannot guarantee the accuracy of measurement under dilution in the dynamic range of unknown samples. The ...
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IPC IPC(8): G16B40/30
Inventor 程标
Owner 领航医学科技(深圳)有限公司