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A primer set and application for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria

A gram-positive bacteria, drug-resistant gene technology, applied in recombinant DNA technology, biochemical equipment and methods, and microbial determination/examination, etc., can solve the problems of difficult treatment of infectious diseases and limited treatment methods

Active Publication Date: 2022-06-21
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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  • A primer set and application for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria
  • A primer set and application for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria
  • A primer set and application for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria

Examples

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Embodiment 1

[0048] Example 1. Establishment of primer sets for high-throughput amplicon sequencing of Gram-positive bacteria drug resistance genes and amplicon sequencing methods

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

[0050] Referring to the ResFinder library, the more common and widespread Gram-positive bacteria drug resistance genes were selected through statistics and analysis, a total of 39 kinds, respectively, on NCBI (https: / / www.ncbi.nlm.nih.gov / ) Download the gene sequence (see file: AMR20190804.fasta).

[0051] figure 1Flow chart for simple sequencing of high-throughput amplicons. figure 2 Schematic for the construction of the amplicon sequencing library (refer to IonAmpliSeq TM Library Kit 2.0 manual). image 3 Design distribution maps for antibiotic resistance genes.

[0052] Through multiple design, analysis and alignment, the final primer pool was obtained. The amplicon length ran...

Embodiment 2

[0097] Example 2. The application of primer sets in the detection of Gram-positive bacteria resistance genes of high-throughput amplicon sequencing was 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 livestock and poultry and 2 samples of human origin were tested (see Table 8). Both the fecal and environmental samples were obtained from China Agricultural University.

[0100] The metagenomic DNA of the stool and environmental samples shown in Table 8 was extracted, and the metagenomic DNA of each sample was used as a template, and amplified according to the second method of Example 1 to obtain the primer pool 1 multiplex PCR amplification product and the primer pool 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 Ex...

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Abstract

The invention discloses a primer set for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria and its application. The invention provides a set of primers for high-throughput amplicon sequencing of drug-resistant genes of Gram-positive bacteria. The set of primers provided by the present invention is composed of primers shown in sequence 1-sequence 78. The present invention establishes an amplicon sequencing detection and analysis method for 39 common clinical drug resistance genes. This method has technical advantages such as rapidity, high efficiency, high targeting, and strong specificity, and is suitable for the prevalence and differential analysis of multiple drug resistance genes in clinical and animal husbandry.

Description

technical field [0001] The invention belongs to the technical field of microbial gene high-throughput sequencing, in particular to a primer set and application for high-throughput amplicon sequencing of Gram-positive bacteria drug resistance genes. 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, and analyze all microbial composition, microbial diversity and their corresponding functions in environmental samples, and then explore the relationship between microorganisms and microorganisms. Links between the environment, microbes, and hosts. Metagenomics is developed on the basis of microbiology and does not depend on the isolation and culture of microorganisms. It can be used to study natural products in difficult or un...

Claims

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Application Information

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Patent Type & Authority Patents(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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