摘要
为了建立能同时快速鉴别沙门菌、鸭疫里默氏杆菌、巴氏杆菌和大肠杆菌的多重PCR检测方法,参考GenBank中沙门菌invA基因序列、鸭疫里默氏杆菌OmpA基因序列、巴氏杆菌KMT1T基因序列和大肠杆菌PhoA基因序列,分别设计特异性引物,用已分离、鉴定、保存的四种细菌DNA筛选PCR缓冲体系、扩增程序,优化退火温度与引物浓度。结果表明,设计的4对引物能分别能特异性扩增出相应的靶基因,建立的多重PCR方法特异性强、敏感性高,可用于沙门菌、鸭疫里默氏杆菌、巴氏杆菌和大肠杆菌混合感染病例的快速鉴别诊断。
To establish a multiplex PCR assay for the rapid identification of Salmonella enteriditis,Riemerella anatipestifer,Pasteurella multocida and Escherichia coli,specific primers were designed,respectively,which refered to the inv A gene sequence of S.enteriditis,the Omp A gene sequence of R.anatipestifer,the KMT1 T gene sequence of P.multocida and the Pho A gene sequence of E.coli in GenBank.The PCR buffer system and amplification program were screened and the annealing temperature and primer concentration were optimized by four kinds of bacterial DNAs which had been isolated,identified and preserved.The results showed that the designed four pairs of primers were high-efficient when amplified the corresponding target genes specifically,and the established multiplex PCR method was highly specific and sensitive.In conclusion,the multiplex PCR method established in this experiment can be used for rapid differential diagnosis of mixed infections of S.enteriditis,R.anatipestifer,P.multocida and E.coli.
作者
包细明
张益
鲜思美
李婷
刘良林
王现科
饶体宇
吴伯梅
张友
BAO Xi-ming;ZHANG Yi;XIAN Si-mei;LI Ting;LIU Liang-lin;WANG Xian-ke;RAO Ti-yu;WU Bo-mei;ZHANG You(College of Animal Sciences,Guizhou University,Guiyang 550025,China;Research Institute ofA nimal Diseases of Guizhou,Guiyang 550025,China;The Agriculture Bureau of Sansui County,Sansui 556500,China;The Agriculture Committee of Liupanshui City,Liupanshui 553001,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2018年第12期1542-1547,共6页
Chinese Veterinary Science
基金
贵州省农业攻关项目[黔科合支撑(2016)2506号]
贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队项目[黔科合人才团队(2015)4016号]
关键词
沙门菌
鸭疫里默氏杆菌
巴氏杆菌
大肠杆菌
多重PCR
Salmonella enteriditis
Riemerella anatipestifer
Pasteurella multocida
Escherichia coli
multiplex PCR
