基于rpoB基因的阪崎克罗诺杆菌分子检测体系的建立及其应用  

Development of molecular detection system of Cronobacter sakazakii based on the rpoB gene and its application to environment-monitoring

作　　者：周胜利[1] 黄佳[2] 晁爱敏[1] 于海燕[1] 顾卿[1] 韩明春[1] 徐杭英[1] 周斌[1] 

机构地区：[1]浙江省环境监测中心,杭州310015 [2]浙江天元生物药业有限公司,杭州311100

出　　处：《安全与环境学报》2012年第4期158-161,共4页Journal of Safety and Environment

摘　　要：通过生物信息学的方法,利用阪崎克罗诺杆菌及其邻近细菌的rpoB基因序列设计了一对特异性引物ESrpoBF及ESrpoBR。特异性检测表明,该对引物能对阪崎克罗诺杆菌进行有效的扩增,PCR扩增产物为154bp大小的条带,而对其余近缘肠杆菌均无阳性扩增。灵敏度检测显示,该对引物在常规PCR及实时定量PCR的检测极限分别为10 pg/μL和10 fg/μL。进一步利用该对引物对浙江省14个重要饮用水源地进行分子检测。结果表明,在6个饮用水源检测到阪崎克罗诺杆菌,检出率为43%。The present research introduces our work in developing a molecular detection system of Cronobacter Sakazakii based on the rpoB Gene and its application to the environment-monitoring practice. Cronobaeter sakazakii is a human opportunistic pathogen which can cause life-threatening neonatal infections. It is foodborne with the primary source of infection from human inhabitation environment. Therefore, it is of great significance for scientists to detect and identi- fy it. It is for this purpose that we are prompt to come to meet such an emergency and developed a Real-time PCR based system. The specific primers in the method were designed based on the non-con- servative region of rpoB sequences by bioinformatics analysis. We have chosen two Cronobacter sakazakii strains and 12 closely related Enterobacter and Cronobacter strains to evaluate its specificity. The results of research show that Cronobacter sakazakii has a positive reaction to 154 bp amplification while all the other closely related strains just have negative reaction. To evaluate its sensitivity, we have adopted pure Cronobacter sakazakii DNA at the concentrations of 10 pg/μL, 1 pg/μL; 100 fg/μL, 10 fg/μL and 1 fg/μL to determine the limitation of the detection. The results of our research indicate that the Real-time PCR method can at least detect at least 10 fg/gL of the DNA reliably. However, conventional PCR method can only be used to evaluate the sensitivity of the detectable DNA concentration at about 10 pg/μL. In addition, when we characterized the method in terms of its sensitivity and specificity, we also conducted further analysis by using this pair of primers to detect some environmental water samples we had collected from the fourteen important drinking water sources in Zhejiang. The results of our investigation of the sources prove that such kinds of pathogen were able to be detected only in six of the fourteen. And, now, we used again some conventional methods to identify the pathogen further, and, in turn, verified some samples

关 键 词：环境学 阪崎克罗诺杆菌 阪崎肠杆菌 RPOB 实时定量PCR 水源 

分 类 号：X832[环境科学与工程—环境工程]