多重PCR快速检测鉴别牛布鲁氏菌和牛分枝杆菌的研究与应用

Development of a rapid and sensitive assay for the detection of Brucella abortus and Mycobacterium bovis in dairy cows by multiplex PCR

目的利用多重PCR方法建立一种同时快速检测鉴别牛布鲁氏菌和牛分枝杆菌的方法。方法根据牛布鲁氏菌具有种特异性的BCSP-31K基因和牛分枝杆菌23SrRNA,设计并合成了两对分别扩增牛布鲁氏菌和牛分枝杆菌的特异性引物,建立了多重PCR同时快速检测鉴别以上两种病原体的方法。其扩增片段大小牛布鲁氏菌为311bp、牛分枝杆菌为838bp。对所建立的多重PCR方法进行特异性试验和敏感性试验。并应用所建立的方法对临床样品进行检测。结果该多重PCR方法对所有供试的牛布鲁氏菌都能扩增出311bp,结核分枝杆菌都能扩增出838bp目的片段,对其它参试牛的菌株则无311bp和838bp条带,该多重PCR方法对牛布鲁氏菌和牛分枝杆菌的DNA最低检出量为10pg。305份临床样品中10份牛奶样品为牛布鲁氏菌阳性,41份包括36份PPD阳性鼻粘液样品、3份PPD可疑鼻粘液样品和2份PPD阳性牛奶样品为牛分枝杆菌阳性,其余样品均为阴性。

Using multiplex PCR to develop a rapid and sensitive method of detecting Brucella abortus and Mycobacteriurn boris simultaneously in dairy cows, two pairs of primers were designed and synthesized from species-specific BCSP-31K gene of Br. abortus and 23S rRNA gene of M. tuberculosis complex respectively. A multiplex PCR amplification detection plateform for the diagnosis of infections with M. bovia and Br. abortus in bovine milk and nasal secretions was developed, in which the sizes of the amplification fragments for Br. abortus and M. boris were 311 bp and 838 hp respectively. The specificity and sensitivity of the established multiplex PCR assay were determined accordingly, and it was used for detection of organisms in clinical specimens. It was demonstrated that both the 311 bp gene fragment of Br. abortus and the 838 bp gene fragment of M. boris could he detected successfully by this multiplex PCR assay, while there was no corresponding band with other reference strains. The minimal detection amount of DNA extracted from these two pathogens accounted for 10 μg among 305 clinical specimens to be tested, 10 milk specimens were positive for Br. abortus, and 41 specimens including 31 PPD-positive nasal secretion specimens, 3 PPD-suspected nasal secretion specimen and 2 milk samples were positive for M. boris. The others were negative for both Br. abortus and M. bowls.