牛布鲁菌与牛分枝杆菌双重PCR方法的建立及应用  

Establishment and Application of a Multiplex PCR Assay for the Detection of Brucella abortus and/or Mycobacterium bovis

作　　者：潘文[1] 王波[1] 薛红 赵明秋[1] 琚春梅[1] 陈金顶[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广州市动物卫生监督所,广东广州510440

出　　处：《华南农业大学学报》2010年第4期100-103,107,共5页Journal of South China Agricultural University

基　　金：广东省动植物防疫检疫研究专项(粤财农[2009]225号);国家自然科学基金-广东省自然科学基金联合项目(U0631006);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0723)

摘　　要：根据GenBank中已发表的流产布鲁菌种特异的omp25基因序列及牛分枝杆菌特异保守的16S rRNA加工蛋白rimM基因序列设计了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鲁菌Brucella abortus和牛分枝杆菌Mycobacterium bovis的双重PCR方法.对建立的方法进行特异性和敏感性试验,结果显示,在牛布鲁菌和牛分枝杆菌中分别扩增出448、269 bp的特异性目的条带,作为对照的大肠杆菌Escherichia coli、沙门菌Salmo-nella typhimurium、八叠球菌Sarcina lutea、金黄色葡萄球菌Staphylococcus aureus、巴氏杆菌Pasteurella multocida、军团菌Legionella pneumophila、枯草杆菌Bacillus subtilis及溶血性链球菌Streptococcus pneumonia均未扩增出任何条带.牛布鲁菌和牛分枝杆菌的DNA最低检出量均为10 pg;牛奶样品中人工污染的牛布鲁菌和牛分枝杆菌的检测敏感性分别为7.9×103CFU/mL、3 ng/mL.A multiplex PCR assay for the detection of Brucella abortus and Mycobacterium bovis infection simultaneously in bovine milk was established using two pairs of primers designed according to the species-specific omp25 gene of B.abortus and conserved 16S rRNA rimM gene of M.bovis.The multiplex PCR assay amplified unique 448 bp and 269 bp amplicons from B.abortus and M.bovis respectively,while none of 8 other common bacterial species strains revealed any amplification products.Sensitivity of genomic DNA detection of B.abortus and M.bovis were both 10 pg,meanwhile the limit of detection in artificially contaminated milk with B.abortus and M.bovi were as low as 7.9×103CFU/mL and 3 ng/mL,respectively.

关 键 词：牛布鲁菌 牛分枝杆菌 双重PCR 快速鉴别检测 

分 类 号：S852.61[农业科学—基础兽医学]