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Method for activity quantification and host identification of antibiotic resistance gene in environment based on metatranscriptomics and metagenomics

An antibiotic resistance and macrotranscriptome technology, which is applied in the field of activity quantification of novel antibiotic resistance genes and host identification, can solve the problems of inability to quantitatively analyze the activity of resistance genes and difficult to locate genes, etc.

Pending Publication Date: 2021-09-03
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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Problems solved by technology

[0003] In the past, the quantitative detection of resistance gene activity in environmental microbial populations mainly relied on QPCR technology, but this technology is difficult to locate the host of the gene, and cannot quantitatively analyze the activity of resistance gene at the strain level (especially for strains that are difficult to isolate and cultivate in the environment)

Method used

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  • Method for activity quantification and host identification of antibiotic resistance gene in environment based on metatranscriptomics and metagenomics

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 quantitative detection and data analysis method

[0033] (1) Sample collection and nucleic acid extraction

[0034] Environmental water or soil microbial samples were collected, quickly frozen with liquid nitrogen, and stored at -80°C before nucleic acid extraction.

[0035] Total DNA was extracted using the FastDNATM Spin Kit for Soil kit (MP Biomedicals, USA). The quality of the DNA was checked by 1% agarose gel electrophoresis, and the DNA concentration was checked using a Nanopdrop.

[0036] Total RNA was extracted using RNeasy Mini Kit (QIAGEN, USA), and residual genomic DNA was digested using RNase-Free DNaseSet (QIAGEN, USA). The extracted nucleic acid samples were stored at -80°C.

[0037] (2) Metagenome sequencing and metatranscriptome sequencing

[0038] DNA samples were subjected to metagenomic sequencing through the Illumina NovaSeq (PE150) platform, with a sequencing depth of about 10Gb. The sequencing process is briefly described as follo...

Embodiment 2

[0043] Example 2 Quantitative detection and host location of the activity of the novel resistance gene GMC oxidase to chloramphenicol antibiotics

[0044] The invention has been successfully applied to the quantitative detection of the activity of the new resistance gene GMC oxidase gene of chloramphenicol antibiotics, and it is found that the expression of the gene in the bioreactor flora can be increased after adding 120 mg / L chloramphenicol for 7 hours The increase was 11.26 times, which confirmed the feasibility of this method for the quantitative detection of new antibiotic resistance gene activity.

[0045] The aerobic enrichment reactor of chloramphenicol resistant bacteria after long-term cultivation was taken as the research object. The reactor uses the activated sludge of the local sewage treatment plant as the initial bacterial population, and uses the inorganic salt medium (KH 2 PO 4 , 7.0g / L; Na 2 HPO 4 , 0.67g / L; CaCl 2, 0.015g / L; MgSO 4 , 0.097g / L; 30mg / L,...

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PUM

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Abstract

The invention relates to a method for activity quantification of a novel antibiotic resistance gene in an environment based on metatranscriptomics and metagenomics. The method specifically comprises the following steps: 1) extracting total DNA and RNA from an environmental microorganism sample; 2) performing metagenome sequencing on a DNA sample, and performing macro transcriptome sequencing on a RNA sample; and (3) analyzing metagenome sequencing and metatranscriptome sequencing results, identifying the novel antibiotic resistance gene and the host thereof, and quantifying the activity of the novel antibiotic resistance gene. The method disclosed by the invention does not depend on strain isolated culture, and activity quantification and host identification of the antibiotic resistance gene in environmental microorganism samples can be realized by combining metagenome and metatranscriptome methods.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a method for quantifying the activity of a novel antibiotic resistance gene in an environment based on metatranscriptomics and metagenomics and identifying a host. Background technique [0002] Antibiotic resistance gene (ARG) as an environmental pollutant, its spread and spread has become an important issue threatening human health around the world. At present, most of the detections for ARGs are for known ARGs, and the lack of identification and understanding of new ARGs has resulted in the underestimation of ARG groups in the environment. In the past, the quantitative detection of ARG mainly analyzed ARG at the DNA level through PCR and metagenomics, which ignored the expression activity of ARG. Highly actively expressed ARG poses a greater threat to human health, and it is urgent to develop new detection and analysis methods to quantitatively detect the expres...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869G16B20/00G16B30/00G16B35/00
CPCC12Q1/6869G16B20/00G16B30/00G16B35/00C12Q2535/122C12Q2537/165
Inventor 李炳张家禹梁贺彬黄锦雷华新
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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