牛布氏杆菌和副结核分枝杆菌双重PCR检测方法的建立

为快速检测牛布氏杆菌和副结核分枝杆菌,根据GenBank中的布氏杆菌OMP31基因序列(JF918757)及副结核分枝杆菌ISMav2基因序列(AF286339)设计、合成2对特异性引物,通过对PCR条件的优化,建立了快速鉴别检测牛布氏杆菌和副结核分枝杆菌的双重PCR方法。经对建立的方法进行特异性和敏感性试验,结果表明,分别扩增出602、246bp的特异性牛布氏杆菌和副结核分枝杆菌DNA目的条带,作为对照的双芽巴贝斯虫、大肠杆菌、沙门氏菌、弓形虫、链球菌、牛放线菌的DNA及其混合物均未扩增出任何条带。牛布氏杆菌与副结核分枝杆菌的最低检测限分别为1.92和2.51pg;牛肉样品中人工污染的牛布氏杆菌和副结核分枝杆菌的检测敏感性分别为6×104和7×104 CFU/mL。该双病原检测体系的成功建立为牛布氏杆菌病及副结核病的检测、鉴定和流行病学调查提供了有力的技术支持。

Establishment of a duplex PCR assay for the differentiation of bovine Brucella and Mycobacterium paratuberculosis

According to the sequences of specific Brucella gene of outer membrane protein(OMP31) and Mycobacterium paratuberculosis gene of C-2 chromosome(ISMav2) available in the GenBank,two pairs of primers were designed to establish a rapid duplex PCR assay for differentiation of Brucella and M.paratuberculosis.Results showed that the duplex PCR assay possessed a high specificity and sensitivity,with 602 bp and 246 bp amplicons amplified from Brucella and M.paratuberculosis,respectively.And none pro-ducts were found from 7 other strains(Babesia bigemina,Escherichia coli,Salmonella typhimurium,Toxoplasma gondii,Streptococcus,Sphaerotilus bovis,the above DNA mixture).Sensitivity of genomic DNA detection of Brucella and M.paratuberculosis were 1.92 pg and 2.51 pg respectively,with the detection limit in artificially contaminated beef as low as 6×104 CFU/mL and 7×104 CFU/mL,respectively.The duplex PCR assay was successfully constructed which will provide strongly technical support for the detection,identification and epidemiological investigations of bovine brucellosis and paratuberculosis.