Identification quantification and genome visualization method for integron-drug resistance gene cassette in environmental sample

A method integrating second-generation sequencing and PCR with third-generation sequencing technologies constructs an integrase gene database for high-throughput identification and quantification of integrons, addressing underestimation issues and enabling effective control of drug-resistant gene cassettes.

CN119132409AActive Publication Date: 2024-12-13EAST CHINA NORMAL UNIV
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Patent Information

Application Number
CN202411163987.9
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2024-08-23
Publication Date
2024-12-13
Estimated Expiration
2044-08-23

AI Technical Summary

Technical Problem

Existing methods for detecting and quantifying integrons and their drug resistance gene cassettes are limited by low-throughput characterization and underestimation of integrase gene sequences, lacking high-throughput and accurate identification and quantification methods for environmental samples.

Method used

A method combining second-generation metagenomic sequencing, PCR amplification, and third-generation long-read sequencing to construct an integrase gene diversity database, using custom scripts for identification and visualization of integron-resistance gene cassettes, including database construction, PCR amplification, and long-read sequencing for genome visualization.

Benefits of technology

Enables high-throughput identification and quantification of integrons and their drug resistance gene cassettes, providing a comprehensive understanding of environmental resistance pollution and supporting targeted control of high-risk resistant pollutants.

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Abstract

The invention discloses a method for identifying, quantifying and visualizing an integron-drug-resistant gene cassette in an environmental sample. The method comprises the following steps: performing high-throughput identification, quantification and genome visualization on a movable genetic element integron based on three-generation sequencing and a drug-resistant gene mediated by the movable genetic element integron; comprising the following steps: 1) integron identification and quantification: constructing an integrase gene database by applying a'collection-screening-verification 'strategy and applying the strategy to integron identification and quantification; 2) identifying the drug-resistant gene cassette and visualizing the genome, namely obtaining DNA (Deoxyribose Nucleic Acid) by utilizing an optimized variable region gene cassette primer in combination with a PCR (Polymerase Chain Reaction) technology, sequencing by adopting PacBio three-generation full-length amplicons to obtain a long sequence of diversity of the gene cassette, and identifying the type of the drug-resistant gene cassette mediated by the integrator on the basis of a professional database; and carrying out visual representation on the integron-multi-drug-resistant gene cassette array by using a self-defined R script, and carrying out quantification based on short sequence and drug-resistant gene cassette long fragment mapping. The invention provides technical support for monitoring and controlling the development of bacterial drug resistance.
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Citation Information

Patent Citations

  • Rapid screening kit for sulfanilamide drug resistance genes and integrase genes in bacteria and primers

    CN110512009A

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