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Identification of antibiotic resistance genes and potential host bacteria based on pma high-throughput sequencing and picrust

A technology of antibiotic resistance and identification method, applied in the field of identification of emerging genotype pollutants and their host bacteria, can solve the problems of high cost, time-consuming qPCR, and dependence

Active Publication Date: 2022-04-19
BEIJING UNIV OF TECH
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] (1) qPCR is time-consuming, laborious and requires specific plasmid standards (obtained by a series of complicated molecular biological processes), which often limits its use in quantifying all ARGs;
[0006] (2) The throughput of HT-qPCR has been greatly improved compared with qPCR, but it still relies on known ARGs primers, and has certain shortcomings in exploring unknown ARGs in the environment
However, metagenomic sequencing is expensive, and it will cost a lot of money to obtain the occurrence characteristics of ARGs in environmental samples in large-scale time and space

Method used

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  • Identification of antibiotic resistance genes and potential host bacteria based on pma high-throughput sequencing and picrust
  • Identification of antibiotic resistance genes and potential host bacteria based on pma high-throughput sequencing and picrust
  • Identification of antibiotic resistance genes and potential host bacteria based on pma high-throughput sequencing and picrust

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Experimental program
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Effect test

Embodiment 1

[0033] 1. Environmental samples

[0034] The environmental sample is activated sludge taken from the aeration tank of a recycled sewage treatment plant in Beijing.

[0035] 2. PMA treatment of environmental samples

[0036] Before extracting genomic DNA from environmental samples, the activated sludge was pretreated with propidium azide bromide (PMA), which mainly included: PMA reagent preparation and storage; PMA pretreatment; PMA photocrosslinking operation. Among them, the preparation and storage of PMA (2mM) reagent are as follows: Dissolve 1 mg of PMA in 980 μL MilliQ, and store in the refrigerator in the dark (-20°C); Mix a large amount of activated sludge to keep the final concentration of PMA at 3-4μg / mL. After fully mixing, incubate in the dark for 15 minutes, and mix upside down every 4-5 minutes, so that PMA can enter the damaged cells to the maximum extent; PMA light Cross-linking operation: Place the cultivated environmental sample on a blue light cross-linking ...

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Abstract

The invention provides a method for identifying antibiotic resistance genes and their potential host cells based on PMA high-throughput sequencing and PICRUSt, and belongs to the technical field of identification of emerging genotype pollutants and their host bacteria in environmental samples. The method includes the following steps: 1. Using PMA-high-throughput sequencing technology for environmental samples to obtain species annotation information of living microorganisms; 2. Using PICRUSt function prediction software combined with KOs data sets of ARGs to identify ARGs; 3. Potential active hosts of ARGs Bacterial identification, compared with ARGs detection based on metagenomic sequencing, effectively reduces the cost of ARGs detection and improves the comprehensiveness of detection; finally realizes the identification of ARGs and traces the active host bacteria of ARGs. The invention provides a method for solving the problems of high detection cost, incomplete detection and identification of active host bacteria in the existing emerging genotype pollutants in environmental samples.

Description

technical field [0001] The invention belongs to the technical field of identification of emerging genotype pollutants in environmental samples and their host bacteria, and in particular relates to an identification method of antibiotic resistance genes (antibiotic resistance genes, ARGs) and their potential active host bacteria in environmental samples. Background technique [0002] Antibiotics are widely used in the prevention / treatment of human or animal and plant diseases and animal breeding. China is one of the countries that produce and consume the most antibiotics in the world. The metabolic rate of antibiotics in organisms is low (10%-40%), and most of them will be excreted through feces / urine in the form of original drugs or metabolites, posing a huge potential hazard to the environment and human health. Antibiotic-induced bacterial resistance and other issues have attracted widespread attention from all walks of life, and antibiotic resistance genes (Antibiotic Resi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6869
CPCC12Q1/689C12Q1/6869C12Q2535/122Y02A50/30
Inventor 樊晓燕张忠兴高玉玺赵君如徐是龙
Owner BEIJING UNIV OF TECH