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Detection method for helicobacter pylori 23S rRNA (Ribosomal Ribonucleic Acid) gene drug-resistant mutation, and application of detection method

A technology of Helicobacter pylori and a detection method, which is applied in the field of detection of drug resistance mutations of Helicobacter pylori 23SrRNA gene, can solve the problems of inability to detect polyclonal conditions, short read length of second-generation sequencing sequences, and inability to distinguish drug resistance mutations, etc. Drug preparation efficiency and the effect of improving resolution

Inactive Publication Date: 2020-06-19
ACADEMY OF MILITARY MEDICAL SCI
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Problems solved by technology

Although this method may not perform PCR amplification, due to the short read length of the second-generation sequencing sequence, for genes with two or more copies on the genome like 23S, the short sequencing sequence cannot be accurately posted to the reference The correct location of the genome, so accurate double-copy or multi-copy information cannot be obtained
[0006] The above methods directly integrate multiple copies of the 23S rRNA gene for analysis, and cannot distinguish drug-resistant mutations from multiple copies of the 23S rRNA gene, nor can it detect polyclonal conditions in each copy

Method used

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  • Detection method for helicobacter pylori 23S rRNA (Ribosomal Ribonucleic Acid) gene drug-resistant mutation, and application of detection method

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Embodiment 1

[0063] (1) Genomic DNA was extracted from the Helicobacter pylori samples to be detected from gastric biopsies of patients with gastritis in the First Affiliated Hospital of Wenzhou Medical University, and then the library was constructed, and then sequenced using the PromethION sequencer from Oxford Nanopore. After the sequencing was completed The software recommended by the sequencer company was used to split the data to obtain the long-fragment sequencing data of the sample. In order to obtain a more accurate genome and to verify the results of the experimental protocol, the MGI sequencer manufactured by BGI was also used for next-generation sequencing.

[0064] (2) Use the long-segment sequencing data and the second-generation data to assemble the complete genome of the sample. Along the direction of the genome, first search for the specific sequence GGCAAGACGG and GACCC to find the copy of the 23S rRNA gene in the same direction as the genome, and then search in the revers...

Embodiment 2

[0069] (1) Genomic DNA was extracted from the Helicobacter pylori sample (13478) to be detected from the gastric biopsy of another patient with gastritis in the First Affiliated Hospital of Wenzhou Medical University, and then the library was constructed, and then the PromethION sequencer of Oxford Nanopore was used for the sequencing Sequencing, after the sequencing is completed, use the software recommended by the sequencer company to split the data to obtain the long-fragment sequencing data of the sample. In order to obtain a more accurate genome and to verify the results of the experimental protocol, the MGI sequencer manufactured by BGI was also used for next-generation sequencing.

[0070] (2) Use the long-segment sequencing data and the second-generation data to assemble the complete genome of the sample. Along the direction of the genome, first search for the specific sequence GGCAAGACGG and GACCC to find the copy of the 23S rRNA gene in the same direction as the genom...

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Abstract

The invention provides a detection method for helicobacter pylori 23S rRNA (Ribosomal Ribonucleic Acid) gene drug-resistant mutation, and application of the detection method, and relates to the biotechnology field. The detection method comprises the following steps of: obtaining a sequencing sequence of a helicobacter pylori sample and a whole genome sequence of the helicobacter pylori sample, wherein the length of the sequencing sequence is at least 3 kb; then, identifying and positioning each copy of the 23S rRNA gene in a whole genome; screening and distinguishing a specific sequencing sequence which is specifically compared to each copy; and analyzing a drug-resistant mutation locus in the specific sequencing sequence. According to the detection method, a drug-resistant mutation situation of different copies of the 23S rRNA gene in the whole genome of the helicobacter pylori can be independently detected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting drug-resistant mutations of Helicobacter pylori 23S rRNA gene and its application. Background technique [0002] Clarithromycin is a commonly used antibiotic in triple or quadruple therapy for the eradication of Helicobacter pylori, but the resistance of Helicobacter pylori to clarithromycin is increasing. The literature shows that the drug resistance of Helicobacter pylori to clarithromycin is mainly related to point mutations in the V region of the 23S rRNA gene, and the drug resistance mutation types are mainly A2142G, A2143G, and T2182C. There are at least two copies of the 23S rRNA gene in the genome of Helicobacter pylori, and the drug resistance mutations of these two copies may be different. [0003] Currently, there are two main methods for detecting drug-resistant mutations in the 23S rRNA gene of Helicobacter pylori: [0004] First, the PCR-based m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q2600/156
Inventor 刘红洁倪铭伯晓晨
Owner ACADEMY OF MILITARY MEDICAL SCI
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