Whole genome combination targeted amplification library construction method and reagents and pathogen detection method

Abstract

A whole-genome combined targeted amplification library construction method, reagents and pathogen detection method, the library construction method includes: obtaining the adapter ligation product with double-stranded adapters connected to both ends of the whole genome DNA of the sample after fragmentation, which includes the optional Target region and non-target region; use the adapter ligation product as a template, use the first gene-specific primer and the downstream library amplification general primer to perform the first PCR amplification to obtain the first amplification product, and the first gene-specific primer and the target region Binding, the downstream library amplification universal primer binds to the first strand of the double-stranded adapter; the first amplification product is used as a template, and the second gene-specific primer, the downstream library amplification universal primer and the upstream library amplification universal primer are used for the second gene-specific primer. Second PCR amplification to obtain a second amplification product, the second gene-specific primer is combined with the target region, and the upstream library amplification universal primer is combined with the second strand of the double-stranded linker. The method enriches the target area without affecting the non-target area, and has low detection cost and high 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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