牛、羊布鲁菌多重PCR方法的建立与应用

Development and Application of Multiplex PCR in Differentiating B.abortus and B.melitensis

根据布鲁菌属特异性基因BCSP31和布鲁菌种间特异性标志IS711插入序列,设计合成了3对引物,以牛种布鲁菌544A、104M和羊种布鲁菌16M基因组DNA为模板,通过优化反应条件,建立了可同时检测布鲁菌属、牛种布鲁菌和羊种布鲁菌的多重PCR方法。牛种布鲁菌可扩增出301和114bp 2条带,羊种布鲁菌可扩增出301和253bp 2条带,该方法对牛种布鲁菌544A和羊种布鲁菌16M混合DNA模板的最小检出量为100pg,对大肠杆菌O157∶H7、小肠结肠炎耶尔森菌等15种参照菌的核酸扩增结果均为阴性。应用该方法对吉林省某牛场的106份粪便进行检测,虎红平板凝集试验作对照,结果PCR检测9份为阳性,且全为牛种布鲁菌阳性,对应的虎红平板凝集试验也为阳性。结果表明,建立的多重PCR方法具有良好的敏感性和特异性,为布鲁菌病的鉴别诊断提供了一种分子检测工具。

Three pairs of primers were designed according to the specific gene BCSP31 of Brucella genus and insert se-quence IS711.The DNA of B.abortus 544A,B.abortus 104 Mand B.melitensis 16Mstrains were used as the positive control to establish the multiplex PCR assay.Developed a multiplex PCR procedure to identify two major species of the genus Brucella simultaneously.Bands of 301,114 bp could be amplified from strains of B.abortus,and 301,253 bp fromB.melitensis.The multiplex PCR assay could detect as low as 100 pg of mixed B.abortus 544 Aand B.melitensis 16 MDNA.No specific bands could be amplified from control bacteria such as E.coli O157∶H7 and Yersinia enterocolitica.106 cow feces samples were deter-mined by the developed multiplex PCR assay,and total 9 samples displied the B.abortus positive.Rose bengal plate agglutina-tion test was used as controlled trial,and 9 samples were Brucellapositive.The results indicated that the established multiplex PCR assay was highly sensitive and specific.This assay provided a molecular detection tool to differential diagnosis for brucello-sis.