奶牛布鲁氏菌病病原PCR检测方法的建立

ESTABLISHMENT OF THE METHOD TO DETECTE PATHOGEN OF COW BRUCELLOSIS BY POLYMERASE CHAIN REACTION

为了弥补血清学和细菌学方法检测和诊断奶牛布鲁氏菌病存在的不足,根据编码牛种布鲁氏菌31KDa外膜蛋白基因的核苷酸序列设计了1对PCR引物,通过对影响PCR扩增条件的优化,建立了快速检测奶牛布鲁氏菌病病原的PCR方法。特异性检测表明,在同一反应条件下,该引物对引起奶牛布鲁氏菌病的牛种、羊种、猪种布鲁氏菌制备的模板DNA均能扩增出384bp目的基因片段,而对大肠杆菌、金黄色葡萄球菌、马流产沙门氏杆菌、溶血性链球菌、嗜肺巴氏杆菌制备的模板分别进行扩增均呈阴性。敏感性检测显示,最低可检出1.5pg的模板DNA。结果表明:本试验所建立的奶牛布鲁氏菌病病原PCR检测方法具有较高的特异性和敏感性。

For making up the deficiency of detection and diagnosis by serology and bacteriology, the Polymerase Chain Reaction （PCR） to detect Brucellosis was set up by designing the primers according to the sequence of gene encoding the outer membrane protein （31KDa） of Brucella abrtus and the conditions of PCR were optimized. The results of specificity detection showed that the expected gene with 384bp of Brucella was amplified based on the DNA templates of Brucella abrtus, B. melitensis and B. suis in the same conditions, while negative results were obtained using the DNA templates of Escherichia coli, Staphylococcus aureus, Salmonella abortus-equi, Streptococcus sanguis and Pasteurella pneumotropica. The sensitivity of detection indicated that the only 1.5pg DNA of Brucella could be detected. All the results above showed that the method to dectect pathogen of Brucellosis by Polymerase Chain Reaction （PCR） had better specificity and sensitivity.