Second-generation sequencing library building method for identifying bacterial types and drug-resistant types of helicobacter pylori in samples

Abstract

The invention discloses a second-generation sequencing library building method for identifying the bacterial types and drug-resistant types of helicobacter pylori in samples. The method includes the steps of designing and screening primers, wherein according to a to-be-measured target gene, the specific primers are designed; 2, synthesizing universal primer pairs; 3, synthesizing transitional primer pairs; 4, synthesizing library building primer pairs; 5, extracting a genome; 6, in a PCR amplification system, adding all specific primers pairs to conduct a first round of multiple amplification,and recycling and diluting a product to be used as a template of next-round multiple amplification; 7, in the PCR amplification system, adding the universal primer pairs and all the transitional primer pairs to conduct a second round of multiple amplification, and recycling and diluting a product to be used as a template of next-round multiple amplification; 8, in the PCR amplification system, adding the library building primer pairs to conduct a third round of library building amplification, and recycling a product to obtain a prepared library; 9, sequencing the NGS target gene; 10, conducting NGS analysis.

Description

technical field [0001] The invention relates to the field of bacterial typing and drug resistance type testing and analysis of Helicobacter pylori, in particular to a next-generation sequencing library construction method for identifying the bacterial typing and drug resistance type of Helicobacter pylori in a sample. Background technique [0002] At present, there are mainly two ways to construct sequencing libraries, one is to use special kits for library construction, and the other is to use amplicon PCR for library construction. Among them, the advantages of using kits for library construction are high degree of standardization, strong applicability, and can target PCR products of various lengths; but the disadvantages are that reagents are expensive and the operation process is cumbersome. Another kind of amplicon library construction is much simpler and cheaper. However, in the current technology, most methods of amplicon library construction are to amplify and build a...

AI Technical Summary

Problems solved by technology

Among them, the advantages of using kits for library construction are high standardization, strong applicability, and PCR products of various lengths; but the disadvantages are that reagents are expensive and the operation process is cumbersome.

Another kind of amplicon library construction is much simpler and cheaper. However, in the current technology, most methods of amplicon library construction are to amplify and build a single target gene. One-time library construction requires single-gene amplification and repetition or multiplex PCR, but the early stage optimization experiment of multiplex PCR library construction method is complicated and the success rate is very low, and the upper limit of the number of target genes that can be amplified is also very small

Images