利用16S rDNA检测奶牛乳房炎5种病原菌PCR方法的建立及应用  

Detection of Five Bacterial Species in Bovine Mastitis by PCR Based on 16S rDNA Sequence

作　　者：李栋梁[1] 寇亚楠[2] 宋月[2] 魏战勇[2] 王亚宾[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物性食品安全重点实验室,河南郑州450002

出　　处：《河南农业科学》2014年第6期132-136,共5页Journal of Henan Agricultural Sciences

基　　金：河南省重大科技攻关项目(112101110100);郑州市重点实验室项目(114PYFZX509)

摘　　要：为建立快速检测引起奶牛乳房炎5种病原菌的方法,参照GenBank发表的产气荚膜梭菌、乳酸乳球菌、变形杆菌、绿脓杆菌、肠球菌序列,分别根据其16SrDNA保守序列区间设计5对引物,进行PCR扩增。结果显示:这5对引物均能针对自身菌株扩增出目的条带,且具有较强的特异性;产气荚膜梭菌、乳酸乳球菌、变形杆菌、绿脓杆菌、肠球菌菌液DNA的最低检测量分别为3.40、0.89、0.91、3.30、3.60ng/μL;对临床采集的疑似患有乳房炎的奶牛所产的牛奶样品进行检测,结果显示,与传统的细菌分离鉴定相比,新建立的方法具有灵敏、高效的特性。According to 16S rDNA conserved sequence, referring to the GenBank sequence, this test designed five pairs of primers, established a PCR assay for detection of five kinds of bovine mastiffs pathogens. Experimental results showed that：Five pairs of primers for their own strains could amplify positive bands, and possessed high specificity. Sensitivity test showed that the primers were able to detect Clostridium perfringens ,Lactococcus lactis , Proteusbacillus vulgaris , Pseudomonas aeruginosa and Enterococcus bacteria DNA concentration of 3.40,0. 89,0. 91,3.30,3.60 ng/μL. The method was used to detect milk samples which was collected from suspected clinical mastiffs cows. Compared with the traditional bacteria isolation and identification method, the new method had the advantages of sensitivity and efficient. The results show that the new method is sensitive to detect cow mastiffs pathogen and can be used in clinical practice.

关 键 词：奶牛乳房炎 16S rDNA PCR产气荚膜梭菌 乳酸乳球菌 变形杆菌 绿脓杆菌 肠球菌 

分 类 号：S854.43[农业科学—临床兽医学]