Method for detecting new resistance genes in environmental sample based on Nanopore metagenome sequencing

A detection method and resistance gene technology, applied in the field of detection of new resistance genes in environmental samples based on Nanopore metagenomic sequencing, can solve problems such as the spread of drug-resistant bacteria and drug-resistant genes, health threats, etc., and achieve high comparison accuracy , Assembly accurate effect

Pending Publication Date: 2021-10-19
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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Problems solved by technology

The abundance of chloramphenicol in the environment will lead to the generation and spread of drug-resistant bacteria and drug-resistant genes, which will eventually pose a threat to human health

Method used

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  • Method for detecting new resistance genes in environmental sample based on Nanopore metagenome sequencing
  • Method for detecting new resistance genes in environmental sample based on Nanopore metagenome sequencing
  • Method for detecting new resistance genes in environmental sample based on Nanopore metagenome sequencing

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Embodiment 1

[0032] 1. DNA extraction and purification

[0033] The DNA extracted using the kit DNeasy PowerSoil Kit (QIAGEN, USA) has high DNA length and purity (the average length of the DNA fragment is about 15kb, A 260 / 280≈1.8, A 260 / 230≈2.0), and the DNA quality can be Meet the requirements of the Nanopore third-generation sequencing library construction.

[0034] Purification of DNA by 1% agarose gel electrophoresis and gel cutting recovery can increase the average length of DNA samples (that is, remove short fragments and some impurities), thereby improving the quality of library construction.

[0035] The Monarch DNA Gel Extraction Kit (NEB, USA) was used for gel recovery, and the concentration of purified DNA was determined using NanoDrop (ND-One, Thermo Fisher Scientific, USA). For the gel recovery method, refer to the kit instructions.

[0036] 2. Oxford Nanopore Technologies (ONT) third-generation metagenomic sequencing

[0037] Use the Ligation Sequencing Kit 1D (SQK-LSK108)...

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Abstract

The invention provides a method for detecting new resistance genes in an environmental sample based on Nanopore metagenome sequencing. The method comprises the following steps: 1) extracting total DNA from a sample, and purifying the DNA; 2) carrying out metagenome sequencing on the purified DNA; and 3) analyzing the data after metagenome sequencing. The method comprises the following steps: extracting, purifying, purifying and the like to obtain DNA (Deoxyribose Nucleic Acid) meeting the requirements of third-generation Nanopore sequencing; a high-quality long sequence is obtained through sequence assembly, error correction and correction, and the accuracy and reliability of sequence alignment are improved; and the antibiotic resistance gene database is expanded, so that the detection result is more accurate and comprehensive.

Description

technical field [0001] The invention relates to the technical field of biological detection and the field of metagenomic sequencing, in particular to a method for detecting new resistance genes in environmental samples based on Nanopore metagenomic sequencing. Background technique [0002] Currently, the methods for detecting antibiotic resistance genes in environmental samples include bacterial pure culture method, qPCR method, gene chip method and metagenomic method. Bacterial pure culture method is a relatively traditional method, through the cultivation and isolation of bacteria, the use of drug susceptibility tests and whole genome sequencing to detect the resistance genes carried by bacteria, but this method may miss many resistance genes, because many Some bacteria are not culturable. The qPCR method designs primers for the resistance genes of concern, and quantitative detection of resistance genes in samples can be performed by fluorescent quantitative real-time PCR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6869
CPCC12Q1/689C12Q1/6869C12Q2535/122
Inventor 李炳雷华新黄锦梁贺彬
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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