Establishment Of The Real-time Fluorescence Quantitative PCR To Identify B.Abortus, B.Melitensis

This study was designed to establish the accurate and rapid detection method of Brucella spp.Brucella abortus,Brucella melitensis.For Brucella spp. identification,the primers and Taqman probe were designed basing on the BCSP31gene.For Brucella strain identification,these primers and Taqman probes were setted depending on the specific insertion element of an IS711. Standard positive plasmid templates were constructed respectively.And the reaction system and condition of the multiplex real-time fluorescence quantitative PCR were optimized.The standard positive plasmid templates of Brucella spp,Brucella abortus,Brucella melitensis were done ten-fold serial dilutions to make the multiplex real-time fluorescence quantitative PCR standard curve.Assessing the sensitivity, specificity and reproducibility of the multiplex real-time fluorescence quantitative PCR reaction system to establish the real-time fluorescent quantitation PCR method for identifying Brucella abortus,Brucella melitensis.And using this diagnostic method established to detect clinical samples.The results showed the best multiplex real-time fluorescent quantitation PCR reaction system and condition are that the concentration of primer and probe are400nM and200nM respectively,and the annealing temperature is60℃.The diagnostic method have a good specificity because it can specifically detect Brucella, B.abortus, B.melitensis and doesn’t cross-react between Brucella species and other Gram-negative bacteria,especially the Yersinia O:9which has the strongly cross-react with Brucella.In addition,the coefficient of variation of this diagnostic method Cq values are less than0.05,and the detection limit is2.8fg-3.2fg.The results showed that this method has good reliability,stability,and high sensitivity.This diagnostic method was applied to detect clinical samples,and it can fast,accurate identification of B.abortus, and B.melitensis.