The Establishment And Application Of Real-time PCR Method For The Detection Of Brucella Spp.

Objective: As a kind of person and livestock disease,brucellosis can threat the environment and human health seriously.A simple,rapid and reliable detection of Brucella method has important significance for the brucellosis’ s prevention and diagnosis.Real time PCR detection technology is one kind of simple,sensitive and rapid detection method.But in many Brucella real time PCR method for detection of reports,people do not aware of inhibitor will affect the method for sample detection’s accuracy and reliability.This paper establishes a fast,sensitive and reliability real time PCR that containing two reference genes for Brucella,the two reference genes control samples of DNA and PCR detection process,improve accuracy and reliability of test results.Methods: As endogenous reference gene,beta actin gene control the quality of sample DNA;recombinant plasmid for exogenous reference gene,control the quality of PCR amplification process.For Brucella IS711 gene specific,we established a real time PCR method for the sample detection of Rubella,and verify the sensitivity,specificity and stability,and made the standard curve.Used this method that new estsblished and bacteria isolation and culture for the sample detection and,Compare the test results.After that,find the SNP of melitensis bv.3 and Brucella vaccine strain S19/A19,and design the primers and probes.Result:1)established the mammalian beta-actin gene as endogenous reference gene,and designed specific primers and probes.For quality control samples DNA extract;Atlantic salmon beta-globin gene fragment selection established recombinant plasmid as exogenous reference for quality control of PCR amplification.2)Establish a method for the detection of Brucella real time PCR optimization.Establish the Brucella standard,standard curve: only amplified DNA of Brucella,the correlation coefficient R2 : 0.999 Y=-3.34X+42.85;Brucella and exogenous recombinant plasmid amplification,R2:0.997 Y=-3.13X+40.50;simultaneous amplification of Brucella and the internal and external source of reference genes,R2 :0.997(Y=-3.12X+40.9).In the genus Brucella amplification process,effects of exogenous and endogenous reference gene on the test results did not join.The detection method of the detection line up to 12 copy ·μL-1,in the detection of specific,Brucella primer and probe only amplified six classic Brucella,of negative bacteria are nothing but specific amplification.Moreover,this method has good stability.3)The method for testing samples were detected on the 30 copies animal tissue samples,360 animal blood,91 milk samples,including detection of 7 Brucella positive tissue samples,12 copies of Brucella positive blood samples and 3 samples of Brucella positive milk.4)Find Brucella melitensis kind of type 3,Brucella vaccine strain A19/S19 and Rev.1SNPs for gene typing,and design a specific primer and probe.Conclusion: This study established a Brucella real time PCR detection method that can be applied directly,containing two reference genes,and is capable of rapid,sensitive and reliable detection of Brucella in the sample.Then the positive samples were further typing of Brucella SNP gene.