双重PCR检测奶牛隐性乳房炎大肠杆菌与无乳链球菌方法的建立及应用  

Establishment of duplex PCR method for simultaneous detection of Eschertchia coli and Streptococcus agalactiae causing subclinical mastitis of dairy cows

作　　者：曾荣荣 瞿秋红 付玉蓉 郝景锋[2] 

机构地区：[1]吉林农业科技学院动物科技学院,吉林吉林132109 [2]吉林农业科技学院预防兽医学吉林省重点实验室,吉林吉林132109

出　　处：《畜牧与兽医》2017年第10期113-116,共4页Animal Husbandry & Veterinary Medicine

基　　金：吉林省科技厅重点科技攻关项目(20150204024NY);吉林省教育厅项目(吉教科合字[2016]第205号);国家级大学生创新项目(201611439025);吉林省大学生创新项目(2016064);吉林农业科技学院重点学科项目(吉农院合字[2015]第X50号)

摘　　要：为快速检测奶牛隐性乳房炎主要致病菌大肠杆菌和无乳链球菌,分别针对2种致病菌16S-23S rRNA和16S rRNA基因设计2对特异性引物,优化并确立双重PCR反应体系。特异性检测表明,对其他对照菌株未扩增出目的条带;敏感性试验表明,该双重PCR对大肠杆菌、无乳链球菌的最小检测浓度分别为10~2、10~3cfu/mL。同时采用双重PCR与细菌学检查法对送检的96份奶样进行检测,结果双重PCR检出63份大肠杆菌阳性、54份链球菌阳性;细菌学方法检出29份大肠杆菌阳性、37份链球菌阳性。说明本研究建立的双重PCR方法敏感、快速,可用于检测大肠杆菌和无乳链球菌引起的奶牛隐性乳房炎。A duplex PCR method was developed for simultaneous detection of the two major agents of subclinical mastitis of dairy cows, Eschertchia coliandStreptococcus agalactiae. The target sequence ofE. coliwas 16 S to 23 S rRNA spacer region, while that ofS. agalactiaewas16 S rRNA region. The results showed that the method was specific and its sensitivity in detectingE. coliandS. agalactiaewas 10~2cfu/mLand 10~3cfu/mL, respectively. Then 96 milk samples were simultaneously detected by duplex PCR and bacteriological examination method.The results showed 63 positive forE. coliand 54 forS. agalactiaein duplex PCR, while 29 positive forE. coliand 37 forS. agalactiaein bac-teriological methods. These results suggested that the duplex PCR method established in this study was sensitive and rapid for detection ofE.coliandS. agalactiaecausing subclinical mastitis in dairy cows.

关 键 词：大肠杆菌 无乳链球菌 双重PCR 隐性乳房炎 奶牛 

分 类 号：S858.23[农业科学—临床兽医学]