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A kind of multiple PCR detection primer and detection method for detecting four kinds of sheep pathogenic bacteria

A pathogenic and multiplex technology, applied in the field of pathogen detection, can solve the problems of long detection cycle and low sensitivity, and achieve the effects of simple operation, high sensitivity and good specificity.

Active Publication Date: 2018-09-28
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the disadvantages of long detection period and low sensitivity when detecting multiple infections of sheep bacterial diseases in the prior art, the present invention provides a primer and detection method for multiplex PCR detection of four kinds of pathogenic bacteria in sheep

Method used

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  • A kind of multiple PCR detection primer and detection method for detecting four kinds of sheep pathogenic bacteria
  • A kind of multiple PCR detection primer and detection method for detecting four kinds of sheep pathogenic bacteria
  • A kind of multiple PCR detection primer and detection method for detecting four kinds of sheep pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1 Single multiplex PCR reaction

[0063] Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli and Salmonella cultured overnight were used to extract bacterial genomic DNA using a bacterial genomic DNA extraction kit (TIANGEN Company) for PCR amplification. Wherein said Klebsiella pneumoniae is selected from Klebsiella pneumoniae ATCC 700603, Salmonella is selected from ATCC14028 Salmonella typhi, Escherichia coli is selected from ATCC8739 Escherichia coli, and Proteus mirabilis is selected from ATCC12453 Proteus mirabilis.

[0064] Single-plex PCR amplification reaction system (50μL): 5.0μL containing Mg 2+ 10×PCR buffer [750mmol / LTris-HCl(pH8.8), 200mmol / L (NH4) 2 SO 4 , 0.1% Tween 20, 25mmol / L MgCl 2 ], 5.0 μL of 2.5 mmol / L dNTP, 0.5 μL of 5 U / μL Taq polymerase, 1 μL of upstream and downstream primers (each primer concentration is 25 pmol / μL), make up to 50 μL with water.

[0065] The reaction conditions were: pre-denaturation at 94°C for 7min; 30...

Embodiment 2

[0068] Example 2 Multiplex PCR reaction

[0069]Each of the test bacteria was cultured overnight, and the bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (TIANGEN Company) and used for PCR amplification. Among the various test bacteria adopted, wherein said Klebsiella pneumoniae is selected from Klebsiella pneumoniae ATCC 700603, Salmonella is selected from ATCC14028 Salmonella typhi, Escherichia coli is selected from ATCC8739 Escherichia coli, Proteus mirabilis Selected from ATCC12453 Proteus mirabilis. Other bacteria E is Proteus vulgaris ATCC49132, F is Staphylococcus ATCC6538 Staphylococcus aureus, G is Listeria monocytogenes ATCC19114 Listeria monocytogenes, H is Pseudomonas aeruginosa ATCC9027 Pseudomonas aeruginosa.

[0070] The primers were 4 pairs of specific primers designed using Klebsiella pneumoniae type III pilus structural gene, Proteus mirabilis urease synthesis positive regulator R gene, Salmonella invasive antigen conserved g...

Embodiment 3

[0088] Example 3 clinical testing

[0089] Randomly select 20 copies of preserved sheep disease materials, carry out enrichment culture to the tissue fluid in broth culture medium respectively, through enrichment culture, the content of each pathogenic bacteria isolated is all within 10 6 cfu / mL or more. Bacterial genomic DNA was extracted, and the method in Example 2 was used for multiplex PCR amplification. At the same time, routine bacterial isolation and identification were carried out as a control.

[0090] The results of multiplex PCR amplification showed that 13 samples amplified a band of about 600 bp, which showed that it contained Klebsiella pneumoniae, and 10 samples amplified a band of about 380 bp, which showed that it contained Proteus mirabilis; 8 samples A band of about 974 bp was amplified, which showed that it contained Escherichia coli; a band of about 280 bp was amplified from 8 samples, which showed that it contained Salmonella. From the analysis of th...

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Abstract

The invention discloses multi-PCR detection primers for detecting four sheep pathogenic bacteria and a detection method. The primers include Klebsiella pneumoniae upstream and downstream primers, proteus mirabilis upstream and downstream primers, escherichia coli upstream and downstream primers and salmonella upstream and downstream primers. According to the detection method, the primers are adopted to perform multi-PCR amplification reaction with Klebsiella pneumoniae DNA, proteus mirabilis DNA, escherichia coli DNA and salmonella DNA as templates, PCR amplification reaction products are added into loading buffer, the mixture is put into agarose gel containing ethidium bromide, and electrophoresis detection is performed to verify the specificity of the primers. The detection method has the advantages that molecular detection with high sensitivity can be achieved just through conventional instruments, and the detection sensitivity of the method is high; meanwhile, no complex operation system is needed, and detection can be performed under common laboratory conditions; besides, the method is easy and fast to implement, high in sensibility and the like.

Description

technical field [0001] The invention relates to a pathogen detection technology, in particular to multiple PCR detection primers and a multiple PCR detection method for detecting Klebsiella pneumoniae, Proteus mirabilis, Escherichia coli and Salmonella in sheep. Background technique [0002] In recent years, with the development of my country's sheep industry in the direction of scale and intensification and the increasing types of various livestock and poultry diseases, the types of sheep diseases are increasing, and the incidence rate has increased significantly. Kleb's pneumonia Bacteria, Proteus mirabilis, Salmonella, Escherichia coli, Pseudomonas aeruginosa, Brucella and Staphylococcus are the main bacterial pathogens causing sheep disease (Jia Junyuan, 2009), and bacterial multiple infections are becoming more frequent and common , causing huge economic losses to large-scale sheep farms, seriously threatening the sustainable development of sheep breeding industry. Niu Z...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12Q1/689C12Q1/10C12N15/11C12R1/22C12R1/19C12R1/42C12R1/37
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 朱瑞良魏凯胡莉萍钟世勋彭军
Owner SHANDONG AGRICULTURAL UNIVERSITY
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