基于TaqMan探针的Real-timePCR定量检测空肠弯曲杆菌

Application of real-time PCR for quantitative detection of Campylobacter jejuni by TaqMan probe

空肠弯曲杆菌 (Campylobacterjejuni ,C .jijuni)被认为是人类主要食源性病原菌之一。由于空肠弯曲杆菌特殊的生长条件和容易进入不可培养但存活的状态 (Viablebutnonculturable ,VNC) ,所以传统的生化鉴定结果并不一定可靠 ,并且是一项费时而繁琐的工作。核酸检测方法的出现为空肠弯曲杆菌的检测带来了方便。在本文基于LightCycler为平台 ,建立一种基于TaqMan探针的Real_timePCR方法来定量检测空肠弯曲杆菌。用该方法检测时 ,发现所有空肠弯曲杆菌 (11株 )都呈阳性 ,所有其它弯曲菌 (3株 )和其它菌株 (5株 )都是阴性。整个检测过程 6 0min内可以完成 ,检测限度为5CFU ,标准曲线的相关系数为 0 .988。结果表明荧光定量PCR方法既为空肠弯曲杆菌提供了一种特异、敏感、快速和简洁的定量检测方法 ,又为研究空肠弯曲杆菌致病机理提供了一种重要方法。

Campylobacter jejuni is recognized as a leading human food_borne pathogen.Traditional biochemical identification forC.jejuni is not reliable due to special growth requirements and the possibility that this bacterium can enter a viable but nonculturable(VNC) state.Nucleic acid_bassed tests have emerged as a useful alternative to traditional testing.In this article,Based LigntCycler we present fluorescent quantitative PCR assay for quantitative detection of C.jejuni.When this assay was applied,the assay positive for all of the isolates of C.jejuni tested(11 iolates,including type strain ATCC33560) and negative for all other Campylobacter spp(3 isolates) and several other bacteria (5 species tested).The total assa could be completed in 60 min with a detection limit of approximately 2 CFU,and a correlation coefficient was 0.988.Result indicated that fluorescent quantitative detection mehtods private not only a special,sensitive,rapid method for quantitative detection of c.jejuni,but also a important method for researching mechanism pathogenesis of C.jejuni.