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Serotyping method of Actinobacillus pleuropneumoniae, primer set and PCR (polymerase chain reaction) systems

A technique for porcine pleuropneumoniae and Actinobacillus, which is applied in the field of molecular biology, can solve the problems of high cost, small identification range of PCR typing method, long time consumption, etc., and achieves the effects of less reagents, large detection range and high efficiency

Active Publication Date: 2019-10-25
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a multiplex PCR serotyping method, primer combination and PCR system for Actinobacillus pleuropneumoniae. By designing the primers and grouping, the Actinobacillus pleuropneumoniae strain to be tested is identified, and the porcine pleuropneumoniae is overcome. The traditional serological typing method of Actinobacillus is high in cost, time-consuming and has the defects of small identification range of the existing PCR typing method,

Method used

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  • Serotyping method of Actinobacillus pleuropneumoniae, primer set and PCR (polymerase chain reaction) systems
  • Serotyping method of Actinobacillus pleuropneumoniae, primer set and PCR (polymerase chain reaction) systems
  • Serotyping method of Actinobacillus pleuropneumoniae, primer set and PCR (polymerase chain reaction) systems

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Experimental program
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Embodiment 1

[0047] The establishment of embodiment 1 primer grouping system

[0048] In this embodiment, specific primers are designed for each serotype by comparing the conserved gene and the outer lipid membrane protein (OlmA) gene in the capsular synthetic gene cluster of Actinobacillus pleuropneumoniae of serotype 1-15; wherein, Serum type 9 and serotype 11 use the same characteristic gene, which makes the specific sequences of type 9 and type 11 the same, a total of 14 pairs of specific primers, the sequences of which are SEQ ID NO.1-SEQ ID NO.28. In order to make it difficult for the primers in the same PCR system to be reversely complementary to form dimers, and to easily distinguish the size of the amplicon, the specific primers are divided into 4 groups, as follows:

[0049] Table 1 Primer grouping system

[0050]

[0051]

Embodiment 2

[0052] Embodiment 2 Each serotype primer specificity experiment of Actinobacillus pleuropneumoniae strain

[0053] In this embodiment, each serotype-specific band is obtained by the following method:

[0054] 1) Streak the Actinobacillus pleuropneumoniae strains of serotype 1-15 on the TSA medium plate respectively, cultivate a single colony, pick a single colony, boil in a water bath, centrifuge, and obtain the genomic DNA of the strain template;

[0055] 2) Using the genomic DNA obtained in step 1) as a template, respectively using the corresponding specific primers to carry out PCR amplification, the amplification conditions are as follows:

[0056] 10 μL reaction system: 5 μL of 2×Premix Taq, 0.4 μL of each 10 mmol / mL primer, 0.5 μL of template DNA, and then add sterile double distilled water or ultrapure water to make up to 10 μL;

[0057] PCR reaction program: pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 30 seconds, annealing at 57°C for 30 sec...

Embodiment 3

[0060] The multiple PCR serotyping method of embodiment 3 Actinobacillus pleuropneumoniae bacterial strain

[0061] The present embodiment carries out the serotyping of Actinobacillus pleuropneumoniae bacterial strain by following method:

[0062] 1) Streak the Actinobacillus pleuropneumoniae strains of serotype 1-15 on the TSA medium plate respectively, cultivate a single colony, pick a single colony, boil in a water bath, centrifuge, and obtain the genomic DNA of the strain template;

[0063] 2) Using the genomic DNA obtained in step 1) as a template, respectively use the 4 sets of primers provided in Example 1 to carry out PCR amplification, and the amplification conditions are as follows:

[0064] 10 μL reaction system: 5 μL of 2×Premix Taq, 0.4 μL of each 10 mmol / mL primer, 0.5 μL of template DNA, and then add sterile double distilled water or ultrapure water to make up to 10 μL;

[0065] PCR reaction program: pre-denaturation at 95°C for 5 minutes; then denaturation at...

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Abstract

The invention provides a serotyping method of Actinobacillus pleuropneumoniae, a primer set and PCR (polymerase chain reaction) systems. Specific primers are designed respectively to serotypes 1 to 15of Actinobacillus pleuropneumoniae; the specific primers for the serotypes 9 and 11 have identical sequences, including four groups of 14 pairs of specific primers; each group includes 3 to 4 pairs of specific primers; each PCR system comprises a set of primers, and a multiple PCR system is formed; genome DNA of the serotype of Actinobacillus pleuropneumoniae to be tested is used as a template subjected to PCR detection with the four groups of primers; the results are compared with reference bacterial strips. The primers are designed before being grouped and subjected to multiple PCR; 13 serotypes of Actinobacillus pleuropneumoniae can be identified; high accuracy is achieved at the premise of saving serotype identifying cost, and the detection range is wide.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a serotyping method, a primer combination and a PCR system for Actinobacillus pleuropneumoniae. Background technique [0002] Actinobacillus pleuropneumoniae (APP) is a gram-negative bacterium and is the pathogenic bacteria of porcine infectious pleuropneumonia. It can cause infectious pleuropneumonia of pigs and has been found in many countries around the world. The pig industry brings huge losses. Actinobacillus pleuropneumoniae can be divided into NAD-dependent biological type I and NAD-independent biological type II according to whether its growth depends on nicotinamide adenine dinucleotide (NAD). Due to different serological reactions, Actinobacillus pleuropneumoniae can be divided into different serotypes. At present, there are 15 serotypes of Actinobacillus pleuropneumoniae, and biological type I includes serotypes 1-12 and serotype 15, of which Serum types 1 and 5 are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125Y02A50/30
Inventor 周明光徐高原王召贺汤细彪金建云陈章表陈波潘建刚
Owner WUHAN KEQIAN BIOLOGY CO LTD
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