实时荧光环介导恒温扩增检测无乳链球菌方法的建立与评价

Establishment of real-time fluorescence loop-mediated isothermal amplification for detection of Streptococcus agalactiae

目的建立实时荧光环介导恒温扩增(Rea-LAMP)快速检测无乳链球菌的方法,为无乳链球菌的快速检测提供一种新的方法。方法针对无乳链球菌纤连蛋白fbsB基因的6个区域设计6条特异性引物,利用链置换DNA聚合酶(Bst DNA polymerase)恒温反应60min,通过荧光信号实时检测无乳链球菌。结果研究的反应体系在使用引物1时,扩增效果最好;在10株相关菌株的检测中,除无乳链球菌出现很好的阳性外,其余菌株均为阴性;本研究检测无乳链球菌DNA的灵敏度可达到300pg/μl。结论 Rea-LAMP检测无乳链球菌的特异性强、稳定性高、操作简便快速,具有较大的推广应用前景,可用于无乳链球菌的快速检测。

OBJECTIVE To establish the real-time fluorescence loop-mediated isothermal amplification（Rea-LAMP）for rapid detection of Streptococcus agalactiae so as to offer a new method for rapid detection of S.agalactiae.METHODS The fbsB gene was amplified by a set of six specially primers that recognized six distinct sequences of the target.The amplification could be obtained by incubating all of the reagents in a single tube for 60 min of thermostatic reaction with Bst DNA polymerase,and the real-time detection of S.agalactiae was conducted through fluorescent signal.RESULTS The amplification effect was best when the primer was used for 1hour in the reaction system.The detection results showed that of the 10 relevant strains,all showed negative except the S.agalactiae strains presented positive.The sensitivity of this method could reach 300pg/μl in the detection of DNA of the S.agalactiae.CONCLUSIONThe Rea-LAMP is highly specific and stable for detection of the S.agalactiae,and its operation is simple and rapid;it can be used for the rapid detection of the S.agalactiae and has great promotion and application prospect.