Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6

A technology for pleuropneumonia and actinobacillus, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of inaccurate results, time-consuming and labor-intensive, poor sensitivity, etc., achieve strong specificity, reduce the probability of occurrence, The effect of improving accuracy

Active Publication Date: 2015-01-28
INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it takes at least 3-4 days from bacterial isolation and culture to purification and DNA extraction to make a preliminary identification, which is not only time-consuming and laborious, but also ...

Method used

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  • Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6
  • Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6
  • Method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and application of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6

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Embodiment 1

[0047] Embodiment 1: The triple PCR detection method of swine infectious Actinobacillus pleuropneumoniae serotype 2, 3 and 6, comprising the following steps:

[0048] (1) DNA extraction of Actinobacillus pleuropneumoniae serotype 2, 3 and 6 clinical strains:

[0049] ① Scrape the colonies on the surface of the solid medium and collect them into a clean 1.5ml centrifuge tube;

[0050] ② Add 1.5ml of lysate (lysate consists of: 0.15mol / L NaCl, 0.1mol / L NaCl 2 EDTA, lysozyme at 15 mg / ml, pH=8.0).

[0051] ③30min in water bath at 37°C.

[0052] ④ Add 230 μl of absolute ethanol, gently invert the centrifuge tube 10 times to mix evenly, avoid producing a lot of foam, centrifuge briefly, and remove the liquid on the centrifuge cap;

[0053] ⑤ Pour the solution and flocs in step 4 into the DNA adsorption column (placed in the collection tube) with a pipette, and place at room temperature for 2 minutes;

[0054] ⑥Centrifuge at 12000rpm for 1min, discard the collection tube, and put...

Embodiment 2

[0065] Embodiment 2: the reagent kit of triple PCR detection of porcine infectious actinobacillus pleuropneumoniae serotype 2, type 3 and type 6

[0066] The composition of the kit includes: PCR reagent tube, reaction buffer 10×PCR buffer, MgCl 2 , Taq DNA polymerase, dNTPs, gel fuel, loading buffer and three pairs of specific foreign substances, with the DNA of Actinobacillus pleuropneumoniae serotype 2, 3 and 6 as positive controls, and the pig Haemophilus DNA was used as a negative control;

[0067] The specific composition of each ingredient:

[0068] Composition Add volume final concentration 10×PCR buffer 10μl 1×PCR buffer MgCl 2 6μl 25mM dNTPs 1μl 10mM AP 2 f 2.5μl 5mM AP 2 R 2.5μl 5mM AP 3 f 3μl 5mM AP 3 R 3μl 5mM AP 6 f 2μl 5mM AP 6 R 2μl 5mM Taq 0.25μl h 2 o 17.75μl

[0069] Among them, three pairs of primers are DNA fragments synthesized by...

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Abstract

The invention relates to a method and kit for triple PCR detection of porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6 and an application of the porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6. The method comprises the steps: extracting DNA of a clinical porcine infectious pleuropneumonia strain; amplifying a PCR product, to be specific, adding a reaction solution consisting of a 10*PCR buffer solution, MgCl2, TaqDNA polymerase, dNTPs and three pairs of specific foreign matters types 2, 3 and 6 into a PCR reagent tube to extract DNA as a template, with the total volume of the reaction solution being 50 microliters; and finally detecting an amplified product. The method provided by the invention is strong in specificity, high in sensitivity, simple to operate, economic in labor and time, and capable of meeting the requirements for detecting multiple serum type genes at the same time, thus improving the accuracy and specificity in detection. According to the method and the kit provided by the invention, the method and the kit can be used for rapidly and conveniently detecting the porcine infectious actinobacillus pleuropneumonia serum types 2, 3 and 6, can be applied to bacterial identification, disease diagnosis, clinical vaccine screening and molecular epidemiology investigation and analysis, and have a wide market prospect and relatively high economic benefits.

Description

technical field [0001] The invention relates to a PCR detection method for Actinobacillus serotypes, belongs to the field of biotechnology, and in particular to a triple PCR detection method for Actinobacillus pleuropneumoniae serotypes 2, 3 and 6, a kit and the method thereof. application. Background technique [0002] Porcine contact infectious pleuropneumonia, also known as necrotizing pleuropneumonia, is an acute respiratory infectious disease caused by Actinobacillus pleuropneumoniae, characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotizing pleurisy. The case fatality rate of the acute patient is high, and the chronic patient can often endure it. [0003] Pigs of all ages can be infected with porcine contact infectious pleuropneumonia, usually pigs aged 2 to 5 months and weighing 30 to 60 kg are more common. Actinobacillus pleuropneumoniae is a respiratory parasite with high host specificity to pigs. Acute infection can not only be seen...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2600/112C12Q2600/16C12Q2537/143
Inventor 李海利王克领朗利敏徐引弟张立宪朱文豪张青娴游一焦文强许峰郑万录施巧婷宁忠山
Owner INST OF ANIMAL HUSBANDRY & VETERINARY MEDICINE HENAN ACAD OF AGRI SCI
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