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Multiple PCR detection primer groups for dairy cow mammitis pathogenic bacterium and application of multiple PCR detection primer groups

A technology for detecting primers for dairy cow mastitis, applied in the field of microorganisms, can solve the problems of low detection rate, unfavorable accurate detection of the bacteria and timely treatment of diseases, complicated operations, etc., so as to improve detection efficiency and protect milk and its dairy products. safe effect

Inactive Publication Date: 2017-08-08
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional identification methods are mainly through classic methods such as bacterial isolation and biochemical identification, but the classic methods are cumbersome to operate, time-consuming, and low in detection rate, which is very unfavorable for accurate detection of bacteria and timely treatment of diseases

Method used

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  • Multiple PCR detection primer groups for dairy cow mammitis pathogenic bacterium and application of multiple PCR detection primer groups
  • Multiple PCR detection primer groups for dairy cow mammitis pathogenic bacterium and application of multiple PCR detection primer groups
  • Multiple PCR detection primer groups for dairy cow mammitis pathogenic bacterium and application of multiple PCR detection primer groups

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Construction of pMD-18T-pauA recombinant vector

[0038] According to GenBank published Streptococcus agalactiae sip gene (accession number HQ878436), Streptococcus dysgalactiae isp gene (accession number CP002215), Streptococcus uberis pauA gene (accession number KT006567.1), Staphylococcus aureus nuc gene (accession number DQ399678) designed primers to obtain: the upstream primer sip-F-2 and the downstream primer sip-R-2 for amplifying the Streptococcus agalactiae sip gene, the upstream primer isp-F-2 and the downstream primer for amplifying the Streptococcus agalactiae isp gene Primer isp-R-2, upstream primer pauA-F-2 and downstream primer pauA-R-2 for amplifying the pauA gene of Streptococcus uberis, upstream primer nuc-F-2 and downstream primer for amplifying the nuc gene of Staphylococcus aureus nuc-R-2; the nucleotide sequence of the primer is:

[0039]

[0040] Then, using genomic DNA as a template, the full-length sequences of sip, isp, pauA a...

Embodiment 2

[0041] Example 2: Specificity Evaluation Test

[0042] Perform PCR amplification reactions on Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, Salmonella, Streptococcus pneumoniae and Bacillus cereus isolated clinically and preserved by our laboratory,

[0043] The 50μL PCR detection system is:

[0044] Taq enzyme, 3.75U,

[0045] 10× PCR buffer, 5 μL,

[0046] MgCl 2 , the concentration is 1.5mmol / L,

[0047] dNTP, the concentration is 200μmol / L,

[0048] sip-F-1 and sip-R-1 primers, each with a concentration of 0.05 μmol / L,

[0049] isp-F-1 and isp-R-1 primers, the respective concentrations are 0.06 μmol / L,

[0050] pauA-F-1 and pauA-R-1 primers, each with a concentration of 0.16 μmol / L,

[0051] nuc-F-1 and nuc-R-1 primers, each with a concentration of 0.16 μmol / L,

[0052] Sample DNA template 50ng,

[0053] wxya 2 O to make up to 50 μL;

[0054] The conditions of PCR were: 95°C for 5 min; 35 cycle...

Embodiment 3

[0060] Example 3: Sensitivity Evaluation Test

[0061] Extract the recombinant vector constructed in Example 1, sterile ddH 2 O was diluted in a 10-fold gradient, and the copy number was taken as 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 The 7 concentration gradients are respectively templates, set sterile ddH 2 O is a negative control. The PCR reaction system is the same as in Example 2. 1.5% concentration of agarose gel electrophoresis to detect PCR amplification products.

[0062] The results of the susceptibility assessment test of Streptococcus agalactiae were as follows: figure 2 As shown, the lanes 1-7 in the figure are respectively: the copy number is 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 recombinant vector, lane 8 is sterile ddH 2 O negative control, lane 9 is positive control of four recombinant vectors, lane M: DL1,000 DNA Marker. It can be seen from the figure that the detection limit of PCR is 10 5 copy number.

[0063] For the results of t...

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Abstract

The invention discloses multiple PCR detection primer groups for a dairy cow mammitis pathogenic bacterium and application of the multiple PCR detection primer groups. The detection primer groups comprise an upstream primer sip-F-1 and a downstream primer sip-R-1 which are used for detecting streptococcus agalactiae sip genes, an upstream primer isp-F-1 and a downstream primer isp-R-1 which are used for detecting streptococcus dysgalactiae isp genes, an upstream primer pauA-F-1 and a downstream primer pauA-R-1 which are used for detecting streptococcus uberis pauA genes, and an upstream primer nuc-F-1 and a downstream primer nuc-R-1 which are used for detecting staphylococcus aureus nuc genes. According to the application, the primer groups are applied to multiple PCR detection methods for the dairy cow mammitis pathogenic bacterium; and the method is short in detection time, low in cost, high in specificity and simple in result determination.

Description

technical field [0001] The invention relates to a multiplex PCR detection primer set for cow mastitis pathogenic bacteria and an application thereof, belonging to the technical field of microbes. Background technique [0002] Milk is one of the oldest natural beverages, known as "white blood", rich in minerals, and has high nutritional value. With the development of China's economy and the continuous improvement of people's living standards, the consumption demand for dairy products has grown rapidly. Cow mastitis is one of the main diseases restricting the development of China's dairy farming industry. It not only reduces milk production and causes economic losses, but also greatly reduces the quality of milk. [0003] Bouine Mastitis is an inflammation of the udder caused by various etiologies. The incidence of dairy cow mastitis in my country is very high. According to reports, the detection rate of dairy cow mastitis in most areas of China is between 40% and 65%. Ther...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/46C12R1/445
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143
Inventor 袁建丰卞国志刁巧虹王娟王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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