Single-molecule tag primer applied to multiple PCR amplification of absolute quantitative high-throughput sequencing and application of single-molecule tag primer

Abstract

The invention provides a single-molecule tag primer applied to multiple PCR amplification of absolute quantitative high-throughput sequencing and application of the single-molecule tag primer. The single-molecule tag primer comprises a 5' end modification group, a nucleotide fragment and a 3' end modification group, wherein the 5' end modification group comprises a UMI molecular tag and a universal primer joint; the UMI molecular tag is connected with the 5' end of the nucleotide fragment; the universal primer joint is connected with the 5' end of the UMI molecular tag; the nucleotide sequence is matched with a target sequence, and is used for amplifying the target sequence; the 3' end modification group comprises an RNA residue, a DNA protection basic group and a blocking group; the RNA residue is connected with the 3' end of the nucleotide fragment; the DNA protection basic group is connected with the RNA residue; and the blocking group is connected with the DNA protection basic group. The single-molecule tag primer has good specificity, high amplification efficiency and wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a single-molecule index primer applied to multiplex PCR amplification of absolute quantitative high-throughput sequencing and an application thereof. Background technique [0002] There are two main methods for the targeted capture of pathogenic microorganisms: one is the hybrid capture method, and the other is the multiplex PCR amplification method. Multiplex PCR amplification refers to simultaneous amplification of more than two amplicons in one reaction system. The main process of the existing multiplex PCR amplification method is to design multiple primers for the common and clear pathogenic bacteria in a lesion sample, and use the designed specific multiplex primers to perform the first round of multiplex amplification in one or more reaction tubes. PCR amplification enriches the target region sequence of the target pathogenic bacteria; then the ampl...

AI Technical Summary

Problems solved by technology

[0007] In view of the deficiencies and actual needs of the existing technology, the present invention provides a single-molecule index primer and its application for multiplex PCR amplification of absolute quantitative high-throughput sequencing. The final amplification reaction system can effectively solve the problems of dimer formation and non-specific amplification, and can realize accurate quantification of the original amplification template, with excellent stability and amplification uniformity, and has broad application prospects

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