A method for detecting microbial target sequences based on single-primer probe capture

Abstract

A method for detecting a microbial target sequence based on single-primer probe capture, comprising: fragmenting a DNA sample containing a microbial target sequence, and then performing end repair; connecting the end-repaired DNA fragments to a joint; using a single-primer probe to connect with the target The probe binding site of the sequence is annealed and extended for target sequence capture, wherein the probe binding site is located upstream or downstream of the target sequence, and the single-primer probe includes a probe sequence portion and a common sequence located at the 5' end of the probe sequence portion Part; the captured target sequence was amplified using a pair of universal PCR primers; the amplified product was subjected to sequencing analysis. The method of the invention eliminates the need for elution to separate the target fragment, simplifies the operation steps, reduces unnecessary loss of the target fragment, and the hybridization time is relatively short, which is suitable for rapid detection requirements; a single primer probe can be captured, reducing the number of early probes The complexity of the design; the use of common primers during amplification removes the heterogeneity caused by the enrichment of different primers.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a method for detecting microorganism target sequences based on single-primer probe capture. Background technique [0002] With the development of sequencing technology, the advantages of next-generation sequencing (NGS) detection methods in various fields have gradually become prominent. Compared with traditional research methods, it is fast, time-saving and accurate in results. It is widely used in various research fields. However, due to the relatively long cycle of current whole genome sequencing (WGS), the relatively high cost of sequencing still limits the application scope of WGS. The application of target sequence capture technology just solves the problems of long time and high cost of WGS sequencing. At the same time, the capture and enrichment of specific target sequences reduces the proportion of unneeded data in the sequencing results and increases th...

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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