Whole genome combined targeted amplification library building method, reagent and pathogen detection method

Abstract

The invention relates to a whole genome combined targeted amplification library building method, a reagent and a pathogen detection method. The library building method comprises the following steps: obtaining a linker connection product of which two ends are connected with double linkers after whole genome DNA fragmentation of a sample, wherein the linker connection product comprises an optional target area and a non-target area; taking the linker connection product as a template, adopting a first gene specific primer and a downstream library amplification universal primer to carry out first PCR amplification to obtain a first amplification product, combining the first gene specific primer with the target region, and combining the downstream library amplification universal primer with a first chain of the double linker; and taking the first amplification product as a template, performing second PCR amplification by adopting a second gene specific primer, a downstream library amplification universal primer and an upstream library amplification universal primer to obtain a second amplification product, combining the second gene specific primer with the target region, and combining the upstream library amplification universal primer with a second chain of the double-chain connector. The method enriches the target area, does not affect the non-target area, and is low in detection cost and high in efficiency.

Description

technical field [0001] The invention relates to the technical field of sequencing detection, in particular to a whole genome combined with targeted amplification library construction method, reagents and pathogen detection method. Background technique [0002] The pathogen detection project is a technology often used by hospitals, CDCs, entry-exit inspection and quarantine bureaus, customs and other units. At present, routine pathogen detection generally uses methods such as real-time fluorescent quantitative PCR technology, bacterial isolation and culture, and immunohistochemistry to detect unknown pathogens. Such technologies often have insufficient detection sensitivity, and cannot detect new mutations and unknown pathogens, resulting in missed detection. , causing a false negative. The next-generation sequencing technology is favored by testing institutions due to its large throughput, high detection sensitivity, and ability to simultaneously detect known and unknown pa...

AI Technical Summary

Problems solved by technology

On the other hand, some samples, such as stool samples, have very complex components and a large content of bacteria, and the detection sensitivity based on fluorescent quantitative PCR technology is not enough, resulting in the missed detection of pathogens

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