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Method for realizing multi-site sequence molecular typing on haemophilus parasuis

A technology for molecular typing of Haemophilus parasuis, applied in the field of multi-locus sequence molecular typing of Haemophilus parasuis, can solve the problem of no prevalence of Haemophilus parasuis disease, easy drug resistance, and lack of immune crossover sexual issues

Inactive Publication Date: 2018-10-12
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, Haemophilus parasuis is very easy to produce drug resistance, and there are many serotypes, and there is a lack of cross-immunity between each serotype, so there is still no good way to control the prevalence of Haemophilus parasuis disease

Method used

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  • Method for realizing multi-site sequence molecular typing on haemophilus parasuis
  • Method for realizing multi-site sequence molecular typing on haemophilus parasuis
  • Method for realizing multi-site sequence molecular typing on haemophilus parasuis

Examples

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Embodiment 1

[0047] 1. Extraction of bacterial genomic DNA

[0048] Follow the instructions of the DNA extraction kit to extract bacterial genomic DNA;

[0049] 2. Housekeeping gene determination

[0050] Screen 7 bacteria to test housekeeping genes rpoB, atpD, g3pd, infB, mdh, _6pgd, frdB;

[0051] 3. Housekeeping gene amplification, detection and sequencing

[0052] The designed primer pair combinations are shown in Table 1. As shown in Table 1, the name, function and primer sequence length information of 7 pairs of housekeeping genes and optimized PCR reaction system and conditions are used to amplify housekeeping genes;

[0053] Table 1

[0054]

[0055] The 25μL reaction system for PCR amplification of 7 housekeeping genes is as follows:

[0056]

[0057]

[0058] PCR reaction conditions are: pre-denaturation temperature 95°C, time 5min; denaturation temperature 95°C denaturation time, 30s; annealing temperature of each housekeeping gene, as shown in Table 2, time 30s; extension temperature 72°C, ...

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Abstract

The invention discloses a method for realizing multi-site sequence molecular typing on haemophilus parasuis. According to the method, the operation technology of carrying out molecular typing on the haemophilus parasuis by adopting the multi-site sequence typing (MLST) method is disclosed for the first time. In the method, the genome DNA of the pathogenic bacteria is extracted, 7 house-keeping genes including rpoB, atpD, g3pd, infB, mdh, _6pgd and frdB are adopted for carrying out screening, amplification, detection and sequencing, CExpress and DNAMAN software is adopted for carrying out splicing, multi-sequence alignment and analysis on the sequencing result, and then the sequence type (ST) of the strain is obtained through an on-line haemophilus parasuis MLST typing database. The amplification primers used by the typing technology are adopted for the first time, the PCR reaction system and conditions automatically optimized, and the technique process can be used for the classification identification and genetic diversity research of haemophilus parasuis.

Description

Technical field [0001] The invention relates to a molecular typing technology of animal pathogens, in particular to a method for molecular typing of Haemophilus parasuis multi-site sequence. Background technique [0002] Haemophilus parasuis strains (HPS) is a short gram-negative bacterium belonging to the Pasteuraceae family, which mainly harms piglets and young pigs. The pathogen is a common bacteria in the upper respiratory tract of pigs. Under certain conditions, it can invade the body and cause serious systemic diseases. Its main characteristics are fibrinous polyserositis, arthritis and meningitis. The pathogen The disease caused is also called "Gera's disease". [0003] At present, Haemophilus parasuis is widespread in my country and even the world, causing serious economic losses to the pig industry. It is one of the world's diseases and has attracted widespread attention. In addition, Haemophilus parasuis is very easy to develop drug resistance, and there are many seroty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12R1/21
CPCC12Q1/689
Inventor 闫鹤万锈琳李春玲
Owner SOUTH CHINA UNIV OF TECH
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