Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis

A technology for Haemophilus suis and serotyping, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. It can solve problems such as backwardness and impracticality, and achieve high accuracy and short time consumption. , good specific effect

Active Publication Date: 2015-04-29
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no such molecular typing method clinically, so it is urgent to establish a comprehensive detection system that can detect 15 serotypes of Haemophilus parasuis conveniently, accurately, and with a high typing rate to replace the traditional Outdated, impractical, and difficult-to-implement serotyping methods

Method used

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  • Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis
  • Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis
  • Serotyping PCR (polymerase chain reaction) method for haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Design of PCR primers

[0042] Compare the conserved genes on the CPS gene cluster of Haemophilus parasuis, and analyze and compare the differences in the conserved genes among the serotypes, design multiple pairs of primers for each serotype, and compare their specificity, sensitivity and other factors through a large number of experiments Finally, a pair of primers with good specificity and high sensitivity are determined for each serotype as identification PCR primers. The sequences of the PCR primers are shown in Table 1, and the sequences of the PCR primers are shown in SEQ ID NO.1-SEQ ID NO.30.

[0043] Table 1 The sequences of PCR primers for each serotype

[0044]

[0045]

Embodiment 2

[0046] Embodiment 2: the establishment of serotyping PCR method

[0047] PCR amplification: Using the genomic DNA of the reference strains of 15 serotypes of Haemophilus parasuis as a template and sterile double-distilled water as a negative control, perform PCR amplification on the 15 pairs of primers designed above.

[0048] All PCR amplifications use a 50 μL reaction system: 25 μL of 2×Taq PCR Master Mix, 1 μL of each primer (10 mmol / mL), 1 μL of template DNA, and then add sterile double distilled water or ultrapure water to make up to 50 μL.

[0049] The PCR reaction procedure is as follows: the system is pre-denatured first, and then undergoes cycles of denaturation-annealing-extension in sequence. Wherein, the pre-denaturation temperature is 95° C., and the pre-denaturation is 5 minutes. In the cycle, the denaturation temperature is 94°C, denaturation is 45s, annealing temperature is 59°C, annealing is 30s, extension temperature is 72°C, extension is 30s, and the numb...

Embodiment 3

[0051] Embodiment 3: the specificity test of serotyping PCR method

[0052] In order to identify the specificity of the serotyping PCR method provided by the present invention to a single serotype, the typing primers of each Haemophilus parasuis were subjected to PCR reaction with the genomic DNA of other 14 serotype strains. Test results such as Figure 2-16 As shown, the above results confirmed that the various types of PCR primers did not cross-react with the other 14 serotypes.

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Abstract

The invention discloses a PCR (polymerase chain reaction) method for designing multiple pairs of primers for 15 serum types by comparing conserved genes on a capsule polysacharide locus of haemophilus parasuis and analyzing conserved gene difference among the serum types, screening a pair of primers as serotyping PCR primers and establishing and identifying the 15 serum types of the haemophilus parasuis. The invention provides the serotyping PCR method for the haemophilus parasuis, which is high in accuracy, high in sensitivity and high in specificity.

Description

technical field [0001] The invention relates to a serotyping method, in particular to a serotyping method for Haemophilus parasuis. Background technique [0002] Haemophilus parasuis (HPS) can cause polyserositis, arthritis and meningitis in pigs. With the development of the pig industry in the world, the disease has become a disease that affects the pig industry worldwide. important bacterial disease. Haemophilus parasuis can affect young pigs from 2 weeks to 4 months. It mainly occurs after weaning and nursery. It is usually seen in pigs aged 5 to 8 weeks. The incidence rate is generally 10% to 15%. The mortality rate can reach 50%. The main clinical symptoms were cough, dyspnea, emaciation, lameness, and rough coat; the main necropsy lesions were fibrinous pleurisy, pericarditis, peritonitis, arthritis, and meningitis. In addition, Haemophilus parasuis can also cause sepsis and may leave sequelae after acute infection, namely abortion in sows and chronic lameness in bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/21
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 贾爱卿刘琪王悦芸周如月安欣宇王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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