实时荧光PCR快速检测鼠疫耶尔森菌方法的建立  

Method establish of real time fluorescent PCR rapid detecting Yersinia pestis

作　　者：陈金超[1] 高敏[1] 鲁立[1] 王丽[1] 徐立风[1] 刘涤瑕[1] 董凤珍[1] 唐德羽[1] 

机构地区：[1]解放军第532医院,安徽黄山245041

出　　处：《现代中西医结合杂志》2010年第2期156-158,共3页Modern Journal of Integrated Traditional Chinese and Western Medicine

摘　　要：目的建立一种能快速检测鼠疫耶尔森菌的实时荧光PCR方法。方法以鼠疫耶尔森菌pla毒力基因的保守区为靶区域设计特异引物和TaqMan荧光探针,探针的5’端标记荧光报告基团FAM,3’端标记荧光淬灭基团TAMRA;建立一种能快速检测样本中鼠疫耶尔森菌的实时荧光PCR方法,并对方法的特异性和灵敏度进行评价。结果该实时荧光PCR方法只对鼠疫耶尔森菌进行特异扩增,同种属的小肠结肠炎耶尔森菌以及伤寒杆菌、肺炎衣原体、嗜肺军团杆菌其他症状相似病原体均无扩增;该方法在65 min内即可完成检测,操作方便快速,对菌悬液最低可检测至10个菌体。结论本研究建立的实时荧光PCR检测鼠疫耶尔森菌方法不仅能实现对鼠疫杆菌的快速检测,还可为鼠疫耶尔森菌引发疫情的监控和溯源提供参考。Objective It is to establish a real time fluorescent PCR method for rapid detection of Yersinia pestis.Methods The conservative area of pla virulence gene in Yersinia pestis was as target area to design specific primer and TaqMan fluorescent probe.The 5＇ end of probe marked fluorescent report group FAM and the 3＇ end marked fluorescent quench group TAMRA.A real time PCR method for rapid detection of Yersinia pestis was established.The specificity and sensitivity of the method were evaluated.Results The real time fluorescent PCR method has specificity amplification action only for Yersinia pestis,but has no amplification action for the congeneric Yersinia enterocolitica,Salmonella typhi,Chlamydia pneumoniae,Legionella pneumophila and some other pathogens of similar symptom.The method could be completed in 65 min and the procedure was convenient and quick.The minimal detection limit was approximated 10 cells.Conclusion The real time fluorescent PCR method established in this study not only can accomplish the quick detection for Bacillus pestis bubonicae,but also can provide reference for monitoring and trace Status of the disease initiated by Yersinia pestis to its source.

关 键 词：鼠疫耶尔森菌 TaqMan实时荧光PCR 快速检测 pla基因 

分 类 号：R-331[医药卫生]