奶牛乳房炎3种主要致病菌三重PCR检测方法的建立  

Development of a Triplex PCR Assay for Detection of Three Major Bovine Mastitis Pathogens

作　　者：陈亚明[1] 杜玉兰[1] 聂培[1] 任怡静[1] 贺婷[1] 杜向宏[1] 杨蓉[1] 左宗辉[1] 何宝祥[1] 

机构地区：[1]广西大学动物科学技术学院,广西南宁530001

出　　处：《中国动物检疫》2014年第7期82-85,共4页China Animal Health Inspection

基　　金：教改项目"广西高等学校动物医学优势专业及课程一体化建设"(编号:GXTSZY141);国家自然科学基金(项目批准号:31260631)

摘　　要：金黄色葡萄球菌、大肠杆菌和蜡样芽孢杆菌是引起奶牛乳房炎的三种常见致病菌,本研究根据金黄色葡萄球菌的nuc基因、大肠杆菌的16S-23SrRNA基因和蜡样芽孢杆菌的hblA基因,设计合成3对特异性引物,通过单纯PCR和多重PCR体系和条件优化,建立奶牛乳房炎主要致病菌三重PCR检测方法,并对50份临床乳房炎乳样分别用传统细菌学培养法和三重PCR方法进行对比检测。结果:建立的三重PCR方法能对同一样品中的3种病原菌以及与沙门氏菌等其他6种细菌的DNA模板进行扩增,均无交叉反应。对金黄色葡萄球菌、大肠杆菌和蜡样芽孢杆菌的检测灵敏度分别为0.1pg/uL、0.01pg/uL、0.1pg/uL。该PCR方法对50份临床乳房炎乳样检测结果,表面该方法的检出率比传统培养法的高。Staphylococcus aureus,Escherichia coli and Bacillus cereus are three common pathogens dairy cow mastitis. Three pairs of specifi c oligonucleotide primers were designed according to the DNA sequences of gene nuc in Staphylococcus aureus,gene 16S-23SrRNA in Escherichia coli and gene hb1A in Bacillus cereus,and a triplex polymerase chain reaction（PCR）assay was developed by improving the systems and procedures of three single PCRs and multiplex PCR. The triplex PCR and traditional bacterial cultivation were evaluated by examining 50 clinical mastitis milk samples. The result showed no cross reaction was detected by the triplex PCR among these 3 bacteria and the other common bacteria. The sensitivity of triplex PCR to Staphylococcus aureus,Escherichia coli and Bacillus cereus was 0.1pg/uL、0.01pg/uL and 0.1pg/uL respectively. The results showed that mutiplex PCR was more sensitive than culture method.

关 键 词：质奶牛 乳房炎 PCR 诊断 致病菌 

分 类 号：S855.1[农业科学—临床兽医学]