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Multiple PCR detection method of swine pathogens

A detection method and technology for swine pathogens, applied in the field of multiplex PCR detection of swine pathogens, can solve the problems of slow growth, false negative results, easy death, etc., and achieve the effects of reducing labor intensity, reducing detection costs, and accurate results

Inactive Publication Date: 2019-03-08
TONGREN POLYTECHNIC COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the isolation of pathogenic bacteria is time-consuming and laborious, which is not conducive to taking measures for timely treatment. The sensitivity is not high, and false negative results are prone to occur. Moreover, the in vitro culture conditions of HPS and Mhp are harsh, slow growth, easy to die, and easily lead to missed detection.

Method used

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  • Multiple PCR detection method of swine pathogens
  • Multiple PCR detection method of swine pathogens
  • Multiple PCR detection method of swine pathogens

Examples

Experimental program
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Embodiment 1

[0041] A multiple PCR detection method for pig pathogens, comprising the following steps:

[0042] (1) DNA extraction from the sample to be tested: use a DNA extraction kit to extract 1 mL each of the DNA of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyopneumoniae in diseased pig tissues, and the DNA extraction method is carried out according to the instructions of the DNA extraction kit. , and then placed in a -20°C environment for standby;

[0043] (2) PCR reaction: add the extracted pig tissue DNA into a reaction tube, mix the liquid in the PCR reaction tube with a vortexer and then centrifuge instantly;

[0044] The system of the PCR reaction is: Buffer2.5μL; Mg 2+ 1.5 μL; dNTP 2 μL; SS upstream and downstream primers 0.5 μL; HPS upstream and downstream primers 0.5 μL; Mph upstream and downstream primers 0.5 μL; Taq enzyme 0.25 μL; template 2 μL; sterile deionized water to 25 μL; the primer sequence For: the sequence of the upstream primer for amplifying th...

Embodiment 2

[0050] The difference from Example 1 is that the template for the PCR reaction also includes recombinant plasmids containing the target gene fragments of the three pathogens, one or more combinations of the three pathogenic genes, sterile distilled water, and other conditions remain unchanged.

[0051] For experimental results, see figure 2 , where M is DL2000marker; lane1 is a positive control: the template contains recombinant plasmids of three kinds of pathogenic target gene fragments; lane2 is a negative control: the template is sterile distilled water; lane3 and 19 are positive for Streptococcus suis; Haemophilus suis, Streptococcus suis, and Mycoplasma hyopneumoniae are positive; lanes 5, 11, and 15 are positive for Haemophilus parasuis, Mycoplasma hyopneumoniae; lanes 6, 9, 10, 13, and 17 are negative; lanes 7, 12 are Streptococcus suis and Mycoplasma hyopneumoniae were positive; lanes 8 and 18 were positive for Haemophilus parasuis; lane 14 was positive for Mycoplasma...

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Abstract

The invention discloses a multiple PCR detection method of swine pathogens. The method includes steps of to-be-detected sample DNA extraction, PCR, PCR amplification, amplification product electrophoresis detection and electrophoresis detection result analysis. A system of PCR comprises 2.5uL of Buffer, 1.5uL of Mg2+, 2uL of dNTP, 0.5uL of SS upstream primer, 0.5uL of downstream primer, 0.5uL of HPS upstream primer, 0.5uL of HPS downstream primer, 0.5uL of Mph upstream primer, 0.5uL of Mph downstream primer, 0.25uL of Taq enzyme, 2uL of template and the balance of sterile deionized water up to25uL. Conventional detection methods of the swine pathogens are relatively time-consuming and labor-consuming, thereby being bad for efficient detection; on the basis of an objective of quickly and accurately diagnosing mixed infectious pathogens, various advantages of multiple PCR detection are utilized to establish a quick, specific and sensitive multiple PCR diagnosis method of the swine pathogens, and quick, accurate and sensitive detection and diagnosis of several diseases are realized.

Description

technical field [0001] The invention belongs to the technical field of gene detection of pathogens, and in particular relates to a multiplex PCR detection method of pig pathogens. Background technique [0002] Porcine respiratory disease is one of the important disease syndromes that endanger the world's pig industry. With the development of the pig industry and the continuous expansion of the breeding scale, the deterioration of the breeding environment and the non-standard breeding scale, the pig respiratory disease is becoming more and more popular and harmful. Seriously, it causes huge economic losses to the world aquaculture industry. Currently, Streptococcus suis (SS), Haemophilus parasuis (HPS) and Mycoplasma hyopneumoniae (Mhp) are the main pathogens that cause respiratory diseases in pigs, respectively causing streptococcal disease and parasuis parasuis. Haematobacillosis and mycoplasma pneumonia lead to acute and chronic inflammation of the respiratory tract of pi...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12R1/46C12R1/35C12R1/10
CPCC12Q1/686C12Q1/689C12Q2537/143
Inventor 曾泽张华琦桂干北綦世金
Owner TONGREN POLYTECHNIC COLLEGE
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