河北省奶牛乳房炎主要致病菌多重PCR检测方法的建立  

Establishment and application of multiple PCR method for detection of main pathogenic bacteria of bovine mastitis in Hebei province

作　　者：夏颖 陈颖彬 马亚宾[2] 王紫燕 刘智慧 高亚桃 赵月兰[1] 秦建华[1] XIA Ying;CHEN Ying-bin;MA Ya-bin;WANG Zi-yan;LIU Zhi-hui;GAO Ya-tao;ZHAO Yuelan;QIN Jian-hua(College of Veterinary Medicine,Agricultural University of Hebei,Baoding,Hebei 071001,China;Animal Husbandry and Breeding Station of Hebei,Shiiazhuang 050000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省畜牧良种工作站,河北石家庄050000

出　　处：《中国兽医学报》2019年第11期2195-2199,共5页Chinese Journal of Veterinary Science

基　　金：河北省现代农业产业技术体系奶牛创新团队建设资助项目(HBCT2018120205)

摘　　要：为同时检测奶牛源金黄色葡萄球菌、无乳链球菌和绿脓杆菌,本试验根据GenBank中3种病原菌的nuc、ef-tu和eta基因保守序列分别合成了3对特异性引物,通过对反应条件、反应体系优化,建立了多重PCR检测方法。特异性试验结果表明,该方法与大肠杆菌、克雷伯杆菌、表皮葡萄球菌、巴氏杆菌无交叉反应,敏感性试验结果表明,多重PCR检测方法对金黄色葡萄球菌、无乳链球菌和绿脓杆菌的检出限为10-3 mg/L。应用该方法对150份临床样品进行检测,结果金黄色葡萄球菌与无乳链球菌混合感染检出率为28.7%(43/150),金黄色葡萄球菌与绿脓杆菌混合感染检出率为14%(21/150),无乳链球菌与绿脓杆菌混合感染检出率为11.3%(17/150),3种菌混合感染检出率为6%(9/150)。应用多重PCR检测结果与分离培养诊断方法结果进行比较,均能检测出样品中的病原菌,2种检测方法的总符合率分别为金黄色葡萄球菌92.6%,无乳链球菌91.3%,绿脓杆菌94.6%。该检测方法对奶牛乳房炎的防控、临床监测及疾病诊治具有重要意义。In order to establish a method for S.aureus,S.agalactiae and P.Aeruginosa detectionin in cows,primers were designed based on the conserved sequence of nuc gene of S.aureus,ef-tu gene of S.agalactiae and eta gene of P.Aeruginosa.After optimizing the reaction conditions and reaction system,a multiple PCR method for simultaneous detection of S.aureus,S.agalactiae and P.Aeruginosa was successfully established.The results showed the methods had no cross-reaction to Escherichia coli,Klebsiella,Staphylococcus epidermidisse and Pasteurella,and could detect 10-3 mg/L for S.aureus,S.agalactiae and P.Aeruginosa,indicating the established methods had high specificity and sensitivity.Clinical detection of 150samples collected from cows with bovine mastitis in Hebei province showed that the co-infection rate of S.aureus 24.9 and S.agalactiae was 28.7%(43/150),and the co-infection rate of S.aureus and P.Aeruginosa was 14%(21/150),and the co-infection rate of S.agalactiae and P.Aeruginos was 11.3%(17/150),which was accordant with the result of soil bacteria isolates.The established method could contribute to the accurate diagnosis,pathogenic surveillance and epidemiological investigation of bovine mastitis.

关 键 词：乳房炎 金黄色葡萄球菌 无乳链球菌 绿脓杆菌 多重PCR 

分 类 号：S857.26[农业科学—临床兽医学]