Method for capturing and detecting microbial target sequence on basis of single primer probe

Abstract

A method for capturing and detecting a microbial target sequence on the basis of a single primer probe comprises the following steps: carrying out fragmentation on a DNA sample which comprises the microbial target sequence, and then carrying out terminal repairing, connecting a DNA fragment with a joint after the terminal of the DNA fragment is repaired; the single primer probe and a probe bindingsite of a target sequence are used for annealing and extend to capture the target sequence, wherein the probe binding site is positioned at the upstream or the downstream of the target sequence, andthe single primer probe comprises a probe sequence portion and a common sequence portion positioned at a 5' terminal of the probe sequence portion; a pair of common PCR primers is used for amplifyingthe captured target sequence; and an amplified product is subjected to sequencing and analysis. By the method, elution is omitted, target fragment separation is carried out, operation steps are simplified, unnecessary loss of target fragments is relieved, hybridization time is relatively short, and rapid detection requirements are met; the single primer probe can be used for capturing immediately,and the complexity of earlier stage probe design is relieved; and during amplification, common primers are used for removing nonuniformity caused by enrichment of different primers.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a method for capturing and detecting a microorganism target sequence based on a single primer probe. Background technique [0002] With the development of sequencing technology, the advantages of next-generation sequencing (NGS) detection methods in various fields are gradually highlighted. Compared with traditional research methods, it is fast, time-saving, and accurate in results. The advantages of accurate and sensitive detection of species DNA sequences have been recognized. It is widely used in various research fields. However, due to the relatively long cycle of whole genome sequencing (WGS), the relatively high cost of sequencing still limits the application range of WGS. The application of target sequence capture technology just solves the problem of long time and high cost of WGS sequencing. At the same time, the capture and enrichment of specific target...

AI Technical Summary

Problems solved by technology

When AmpliSeq technology uses multiplex PCR for enrichment, due to the difference in the amplification efficiency of different primers and the interaction between primers, the enrichment results of different target fragments are not uniform, so it is not suitable for quantitative analysis.

Many PCR amplification cycles are easy to introduce site mutations, which will affect the result judgment

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