Compound PCR typing kit for distinguishing serotypes of eight pig actinobacillus pleuropneumoniae and application thereof

A technology for porcine pleuropneumoniae and actinobacillus, applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of cumbersome operation, low sensitivity, and poor specificity, and achieve high sensitivity and false positives Low, easy-to-operate effect

Active Publication Date: 2018-10-19
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to cumbersome operation, long period, or low se

Method used

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  • Compound PCR typing kit for distinguishing serotypes of eight pig actinobacillus pleuropneumoniae and application thereof
  • Compound PCR typing kit for distinguishing serotypes of eight pig actinobacillus pleuropneumoniae and application thereof
  • Compound PCR typing kit for distinguishing serotypes of eight pig actinobacillus pleuropneumoniae and application thereof

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Embodiment 1

[0030] The method for detecting and distinguishing eight kinds of Actinobacillus pleuropneumoniae serotypes of type 1, type 3, type 4, type 5, type 6, type 10, type 12 and type 14 by using a composite PCR typing kit comprises the following steps:

[0031] (1) Preparation of composite PCR template: ① For clinical tissue samples, wipe and disinfect the tissue samples with alcohol under sterile conditions, remove the outer part with scissors, pick tissue samples with serious lesions of about 1 cm, and use sterile ddH 2 O for washing, boiled for 5-10 minutes, ice-bathed for 3 minutes, centrifuged at 12000r / min for 1 minute, and the supernatant was the template for the multiplex PCR reaction. ② For bacterial strains, use an inoculation loop that has been burned and cooled to room temperature on a sterile operating table to scrape an appropriate amount of freeze-dried powder or bacterial solution from the ampoule of freeze-dried or glycerin-preserved APP and spread it on the TSA petr...

Embodiment 2

[0040] A composite PCR typing kit for distinguishing eight serotypes of Actinobacillus pleuropneumoniae of type 1, type 3, type 4, type 5, type 6, type 10, type 12 and type 14, using the composite PCR The typing kit detects the pig lung tissue submitted by the laboratory, and uses the established multiple PCR method to detect a total of 29 lung tissues submitted by farmers (can be completed within 2 hours), including the following steps:

[0041] (1) Preparation of composite PCR template: For lung tissue samples, wipe and disinfect the tissue samples with alcohol under aseptic conditions, remove the outer part with scissors, pick tissue samples with serious lesions of about 1 cm, and use sterile ddH 2 O was washed, boiled for 5-10min, ice-bathed for 3min, and centrifuged at 12000r / min for 1min, and the supernatant was the template for the compound PCR reaction;

[0042] (2) The composition of the compound PCR reaction system: Add 12.5 μL of PCR mixed enzyme, 0.75 μL of each pr...

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Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to a composite polymerase chain reaction (PCR) typing kit for distinguishing serotypes of eight pig actinobacillus pleuropneumoniae and application thereof. The composite PCR typing kit comprises a PCR test tube, a PCR mixed enzyme, five pairs of specific primers, a positive control, a negative control and ddH2O, wherein the nucleotide sequences of the five pairs of specific primers are shown by SEQ ID NO:1-10. The composite PCR typing kit is used for distinguishing serotypes of eight pig pleuropneumonia actinobacillus of type 1, type 3, type 4, type 5, type 6, type 10, type 12 and type 14, and has high accuracy, good specificity and sensitivity up to a pg level. Sources of samples for identification include isolated strain resources or animal tissues, and a tissue washing liquid or a single colony and a bacterial solution can be directly cracked by boiling water as a template, thus eliminating tedious extraction of bacterial genomes and greatly saving time, manpower and cost.

Description

technical field [0001] The invention belongs to the technical field of biotechnology, in particular to a compound PCR typing kit for distinguishing eight serotypes of Actinobacillus pleuropneumoniae and its application. Background technique [0002] Porcine contagious pleuropneumonia is a serious contagious disease that causes the respiratory system of pigs. It is characterized by acute hemorrhagic fibrinous pleuropneumonia and chronic fibrinous necrotizing pleuropneumonia. Countries and regions where the pig industry is developed have caused huge economic losses to the pig industry. [0003] Porcine infectious pleuropneumonia can infect pigs of all ages, and pigs aged 6 weeks to 6 months are more frequent; and it has obvious seasonality, mostly occurring in April-May and September-November. Sick pigs and pigs with bacteria are the main source of infection of the disease, mostly secondary or concurrent with other diseases, resulting in aggravated clinical symptoms and incre...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2565/125C12Q2537/143
Inventor 袁芳艳田永祥刘泽文刘威周丹娜杨克礼段正赢郭锐高婷
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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