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Multiple PCR detection method for porcine bacteria

A detection method and bacterial technology, applied in the field of multiplex PCR, can solve the problems of inaccurate results, low sensitivity, false negative results, etc., and achieve the effect of reducing false positive rate, high cost and long time

Inactive Publication Date: 2011-03-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it takes at least 2-3 days for bacterial isolation to obtain identification results, which is time-consuming and laborious, which is not conducive to taking measures for timely treatment, and the sensitivity is not high, the results are inaccurate, and false negative results are prone to occur

Method used

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  • Multiple PCR detection method for porcine bacteria
  • Multiple PCR detection method for porcine bacteria
  • Multiple PCR detection method for porcine bacteria

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Experimental program
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Effect test

Embodiment 1

[0019] 1. Collection of specimens: Homogenize 9g of diseased tissue, incubate at 37°C for 4h, then digest with 20g / L proteinase K in a warm bath at 65°C for 3h.

[0020] 2. DNA extraction of the tested specimen: take 1ml of the digested culture and centrifuge at 12000rpm×3min to collect the precipitate, and use ddH 2 O was resuspended and then centrifuged once at 12000rpm×3min, the supernatant was discarded, and an equal volume of 2×TZ and ddH was used for precipitation. 2 Resuspend in O, let stand at -20°C for 45min, put in a boiling water bath for 8min, and immediately place in an ice bath to cool for 10min, at 4°C at 12000rpm×1min, take the supernatant for use.

[0021] 3. Set up the PCR detection kit, which includes:

[0022] (1) PCR reagent tube: the reagent tube contains ddH 2 O 4.87 μl, 10×PCR buffer 2.5 μl, dNTP 2 μl, 6 primers: 5 μl each of A1 and A2, 1.5 μl each of S1 and S2, and 0.2 μl each of P1 and P2. Among them, the 6 primers are DNA fragments synthesized by ...

Embodiment 2

[0038]

[0039]

[0040]

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Abstract

The invention provides a multiple PCR detection method for porcine bacteria. Streptococcus suis type 2, Actinobacillus pleuropneumoniae and pasteurella multocida positive samples are detected by extracting the DNA of test samples, carrying out PCR amplification with PCR testing kit and analyzing the amplified products.The method of the invention can rapidly and accurately detect the samples infected with one or more of the above three bacteria. The template DNA preparation step of the method is simple and the cost is low. The method can exclude the interferences of bacteria and impurity particles, thus greatly improving diagnostic accuracy and reducing false positive rate. The method can also be used for the molecular epidemiological investigation and the efficacy monitoring of Streptococcus suis type 2, Actinobacillus pleuropneumoniae and pasteurella multocida.

Description

technical field [0001] The invention belongs to a biological detection method, and relates to a multiplex PCR method for differential diagnosis of clinical samples with single or multiple mixed infections of three kinds of bacteria, Streptococcus suis type 2, Actinobacillus pleuropneumoniae and Pasteurella multocida. Background technique [0002] Since June 2001, "pig unknown hyperthermia" characterized by high fever, anorexia, and dyspnea began to appear in Anhui, Hunan, Zhejiang and other areas with intensive pig farming in the central and eastern parts of my country. The pathogen of "Porcine Unknown Hyperthermia" is complex, mainly mixed infection and secondary infection of various viruses, bacteria and parasites, including classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine influenza virus, pseudorabies virus, pig Circovirus, Actinobacillus pleuropneumoniae, Streptococcus suis type 2, Haemophilus parasuis, Pasteurella multocida, Mycopl...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 杜爱芳张德福周前进高翔
Owner ZHEJIANG UNIV
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