快速检测牛羊猪种布鲁杆菌多重PCR的建立

Differentiation of Brucella abortus, Bruceila melitensis, and Brucella suis by multiple primers PCR

目的建立一种能同时快速检测并能鉴别牛、羊、猪种布鲁杆菌的多重PCR方法。方法根据IS711插入序列设计1条公共引物和3条牛、羊、猪种布鲁杆菌（544A、16M、1330S）特有序列引物，进行多重PCR反应；选择耶尔森菌0：9、大肠埃希菌0157：H7、鼠伤寒沙门菌47729进行多重PCR反应的特异性检测：倍比稀释定量法观察牛种布鲁杆菌多重PCR反应的敏感性。结果牛、羊、猪种布鲁杆菌多重PCR反应扩增片段产物长度分别为485、731、248bp；耶尔森菌O：9、大肠埃希菌0157：H7、鼠伤寒沙门菌47729加入布鲁杆菌中进行多重PCR反应，扩增结果呈阴性；牛种布鲁杆菌多重PCR反应敏感性为0．0967Pg。结论成功建立快速检测牛、羊、猪种布鲁杆菌多重PCR扩增反应方法，且其特异性、敏感性较好。

Objective To establish a method for rapidly identifying Brucella abortus, Brucella melitensis and Brucella suis by multiple primers PCR. Methods According to BruceUa abortus, Brucella melitensis and Brucella suis IS711 insertion sequences, a public primer and three specific primers（544A, 16M, 1330S） were designed to set up multiplex PCR detection method. Yersinia 0 ： 9, Escherichia coli O157 ： HT, Salmonella typhimurium 47729 were selected to undergo multiple PCR reactions to detect the specificity. The sensitivity of multiple primers PCR of Brucella abortus was detected using multiple proportion dilution method. Results The amplified fragment size of Brucella abortus was 485 bp, that of Brucella melitensis 731 bp, and that of Brucella suis 248 bp, but PCR for the DNA of Yersinia 0 ： 9, Escherichia coli O157 ： H7, Salmonella typhimurium 47729 was negative. A sensitivity of the muhiple primers PCR with Brucella abortus DNA using multiple proportional dilution quantitative method was 0.0967 pg. Conclusions Multiple PCR amplification method for rapidly detecting Brucella abortus, Brucella melitensis and Brucella suis has been successfully established, resulting in good specificity and sensitivity.