TaqM an探针荧光PCR定量检测大肠埃希菌方法研究  

Development of a TaqMan Probe-Based Real-time qPCR Method for the Detection and Enumeration of Escherichia coli

作　　者：胡朝友[1,2] 傅春玲[1] 陆巧荣[2] 张宏斌[2] 

机构地区：[1]苏州大学,江苏苏州215000 [2]江苏省昆山市疾病预防控制中心,江苏昆山215300

出　　处：《现代预防医学》2014年第6期1070-1073,1077,共5页Modern Preventive Medicine

基　　金：江苏省卫生厅预防医学2012年科研课题资助项目(Y2012049);苏州市饮用水安全与水性疾病监测公共服务平台开放实验室开放课题资助项目(SZPT2012001)

摘　　要：目的应用TaqMan探针实时荧光PCR技术，建立大肠埃希菌的快速定量检测方法。方法根据大肠埃希菌的ydiJ基因，设计引物和探针，并分析其特异性和灵敏性。制备大肠埃希菌的质粒DNA标准品，优化qPCR反应检测体系，建立标准曲线，并进行方法学评价。结果该方法有很好的特异性，方法线性范围10～1．0×108拷贝／μl（R2：0．990～0．999）、检测限可达到10拷贝／肛l和定量下限100拷贝／μl。RT—qPCR和培养计数法所得结果具有直线相关关系，r=0．991，P〈0．01。结论RT—qPCR方法可以快速、准确、定量地检测大肠埃希菌。Objective To develope a TaqMan Probe-Based Real-time qPCR Method for the Detection and enumeration of Es- cherichia coll. Methods Specific primers and probes were designed to target the ydiJ gene of Escherichia coll. Moreover, the primer taxonomic specificity and sensitivity were confirmed. We also created a Plasmid DNA Calibration standards containing a segment of the ydiJ gene.The reaction systems and conditions for PCR were optimized. Then Standard curves were created for analy- sis. Results In this study, these primers showed good specificity with all the strains tested. The linearity of quantitative PCR was lin- ear from 10-1.0 × 108copy/μl （R2： 0.990-0.999）. The detection and quantification limits were estimated to be 10 and 100 copy/μA respectively, the results showed that the numbers obtained by qPCR correlated with those measured by plating, r=0.991, P〈0.01. Conclusion The RT-qPCR is a rapid and accurate technique for enumerating Escherichia coli in water samples.

关 键 词：大肠埃希菌 PCR 定量检测 

分 类 号：R446.5[医药卫生—诊断学]