检测牛、羊布鲁菌多重PCR方法的建立与应用研究

ESTABLISHMENT AND APPLICATION OF MULTIPLEX PCR IN DIFFERENTIATING B.abortusand 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3对引物,以牛种布鲁菌A19,羊种M5,猪种S2基因组DNA为模版,建立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,并对方法的扩增效果、特异性、敏感性及适用性进行验证。结果显示,牛种布鲁菌可扩增出222bp和615bp两条带,羊种布鲁菌可扩增出222bp和932bp两条带,该方法对牛种布鲁菌A19和羊种布鲁菌M5混合DNA模板的最小检出量为100pg,对葡萄球菌26001株、大肠杆菌O78等7种参照菌的核酸扩增结果均为阴性。应用该方法对19份布鲁菌血清抗体为阳性牛乳样进行检测,结果均为布鲁菌抗原阳性。

In this research, three pairs of primers were designed according to the specific gene BCSP31 of Brucella genus and insert sequence IS711. The DNA of B. abortus A19, B. melitensis M5 and B. suis S2 trains were used as the positive control to establish the multiplex PCR and the specificity, sensitivity and applicability of this method were verified. Results showed that bands of 222,615 bp could be amplified from strains of B. abortus, and 222,932 bp from B. melitensis. The multiplex PCR assay could detect as low as 100 pg of mixed B. abortus A19 and B. melitensis M5 DNA. No specific bands could be amplified from control bacteria such as E. eoli OTs and Staphylococcus 26001. The method was applied to test 19 milk samples collected from Brucella serum antibody - positive cow, results showed Brucella antigen oositive, indicate multiolex PCR established in this research has a good orosvect.