Method for detecting absolute copy number of fetal free DNA in maternal plasma on basis of digital PCR and kit for detecting absolute copy number of fetal free DNA in maternal plasma on basis of digital PCR

Abstract

The invention provides a method for detecting absolute copy number of fetal free DNA in maternal plasma on basis of digital PCR. The method for detecting the absolute copy number of the fetal free DNAin the maternal plasma on basis of the digital PCR comprises the following steps: obtaining total DNA from maternal peripheral plasma, and selecting gene locus sequences and internal reference gene sequences containing maternal-fetal methylation differences for designing primers and probes; carrying out methylation-sensitive restriction endonuclease digestion; and then, performing digital PCR amplification on the gene locus sequences and the internal reference gene sequences containing the maternal-fetal methylation differences in digital PCR apparatus, detecting fluorescent signals so as toobtain the absolute copy number of the fetal free DNA in the maternal plasma, and excluding false positive interference of maternal free DNA caused by incomplete enzyme digestion. The method for detecting the absolute copy number of the fetal free DNA in the maternal plasma on basis of the digital PCR combines a methylation-sensitive restriction endonuclease digestion-PCR means and a digital PCR platform, and utilizes maternal-fetal methylation difference genes as markers for detecting the absolute copy number of the fetal free DNA in the maternal plasma, so that the method is beneficial for accurately and quickly performing early non-invasive prenatal screening or prenatal diagnosis on pregnant women.

Description

technical field [0001] This application relates to the technical field of digital PCR, in particular to a method and kit for detecting the copy number of fetal free DNA in maternal plasma based on digital PCR. Background technique [0002] In the process of non-invasive prenatal screening and prenatal diagnosis for pregnant women, it is often necessary to know the copy number of fetal cell-free DNA in maternal plasma. [0003] There are currently existing methods for measuring the copy number of fetal cell-free DNA in maternal plasma, including Y chromosome estimation method, SNP-based fetal-specific SNP site method, SNP-based deep targeted sequencing method, SNP-based low-depth sequencing, Based on nucleosome blotting and more. However, these methods in the prior art have certain disadvantages, such as not being suitable for female fetuses, requiring very high sequencing depth, poor accuracy, etc., and it is difficult to accurately obtain the absolute cell-free DNA of the ...

AI Technical Summary

Problems solved by technology

However, these methods in the prior art have certain disadvantages, such as not being suitable for female fetuses, requiring very high sequencing depth, poor accuracy, etc., and it is difficult to accurately obtain the absolute cell-free DNA

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