Primer group for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing and application

A technology of Gram-positive bacteria and drug-resistant genes, applied in the direction of recombinant DNA technology, microbial measurement/testing, biochemical equipment and methods, etc., can solve the problems of difficult treatment of infectious diseases and limited treatment methods

Active Publication Date: 2021-05-28
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In recent years, Gram-positive bacteria are increasingly resistant to various antibiotics, which has caused great difficulties in the clinical treatment of infectious diseases.
Among them, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) are currently one of the important pathogens of nosocomial infections, and their treatment options are very limited.

Method used

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  • Primer group for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing and application
  • Primer group for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing and application
  • Primer group for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1. Establishment of primer set and amplicon sequencing method for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria

[0049] 1. Preparation of primer sets for high-throughput amplicon sequencing of drug-resistant genes in Gram-positive bacteria

[0050] Referring to the ResFinder library, 39 drug-resistant genes that are currently common and widespread in Gram-positive bacteria were selected through statistics and analysis. Download the gene sequence (see file: AMR20190804.fasta).

[0051] figure 1Flowchart for simple sequencing of high-throughput amplicons. figure 2 Construct a schematic for an amplicon sequencing library (see IonAmpliSeq TM Library Kit 2.0 manual). image 3 Design distribution maps for antibiotic resistance genes.

[0052] Through multiple designs, analyzes and comparisons, the final primer pool was obtained. The length of the amplicon ranged from 125bp to 275bp, including two primer pools (primer poo...

Embodiment 2

[0097] Example 2, the application of the primer set of high-throughput amplicon sequencing in the detection of drug-resistant genes of Gram-positive bacteria is carried out according to the second method of Example 1:

[0098] 1. Multiplex PCR to prepare amplicons (2primer pools)

[0099] A total of 20 samples of animal origin and 2 samples of human origin were detected (see Table 8). Both the stool and environmental samples were obtained from China Agricultural University.

[0100] Extract the metagenomic DNA of the feces and environmental samples shown in Table 8, respectively use the metagenomic DNA of each sample as a template, and amplify according to the second method of Example 1 to obtain primer pool 1 multiplex PCR amplification products and primerpool 2 multiplex PCR amplification product.

[0101] Table 8 is the sample information to be tested

[0102]

[0103]

[0104] 2. Construction of amplicon library

[0105] It is the same as Method 2 of Example 1. ...

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Abstract

The invention discloses a primer group for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing and application of the primer group. The invention provides a complete set of primers for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing. The complete set of primers provided by the invention consists of primers as shown in sequences 1-78. The invention discloses an amplicon sequencing detection analysis method for 39 clinical common drug-resistant genes. The method has the technical advantages of rapidness, high efficiency, high targeting, strong specificity and the like, and is suitable for epidemic and difference analysis of various drug-resistant genes in clinic and livestock breeding industry.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing of microbial genes, and in particular relates to a primer set for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria and its application. Background technique [0002] Metagenomics, also known as microbial environmental genomics, metagenomics. By directly extracting the DNA of all microorganisms from environmental samples, constructing a metagenomic library, using metagenomic sequencing to perform high-throughput sequencing of microorganisms, analyzing the composition, diversity and corresponding functions of all microorganisms in environmental samples, and then exploring the relationship between microorganisms and The relationship between environment, microbe and host. Metagenomics, developed on the basis of microbiomics, does not rely on the isolation and culture of microorganisms, and can be used to study natural products and natural produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q2531/113C12Q2537/143C12Q2535/122
Inventor 王少林李一鸣沈张奇沈建忠
Owner CHINA AGRI UNIV
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