文献类型：期刊文献

中文题名：双重实时荧光PCR快速检测O157大肠杆菌的初步研究

英文题名：PRELIMINARY RESEARCH ON RAPID DETECTION OF ESCHERICHIA COLI O157 WITH DUPLEX REAL-TIME FLUORESCENCE PCR ASSAY

作者：张建华[1];卢亦愚[2];程苏云[2];陆群英[2];叶菊莲[2];罗芸[2]

机构：[1]绍兴文理学院医学院;[2]浙江省疾病预防控制中心

年份：2007

卷号：34

期号：14

起止页码：2611

中文期刊名：现代预防医学

外文期刊名：Modern Preventive Medicine

收录：CSTPCD、、北大核心2004、北大核心

基金：浙江省卫生厅医药卫生科学研究基金资助(2005A102)

语种：中文

中文关键词：实时PCR;Taqman探针;O157大肠杆菌;检测

外文关键词：Real-time fluorescence PCR; Taqman--based probe; Enterohemorrhagic E.coli O157; Detection

中文摘要：[目的]建立双重实时PCR体系,实现对O157大肠杆菌rfbE和stx2基因的同步检测。[方法]根据GenBank公布的O157大肠杆菌rfbE和stx2基因序列,应用分子生物学软件设计两对引物和TaqMan探针,并对荧光定量PCR反应条件进行优化,建立实时荧光定量PCR检测O157大肠杆菌的反应体系,并对该法的特异性、敏感性和重复性进行了评价。[结果]所有O157大肠杆菌菌株的检测结果均为阳性,而所有其他菌株检测结果均为阴性;该方法对O157大肠杆菌纯培养的检测范围为100～106cfu/μl,重复性检测的变异系数均小于5%。对模拟污染牛奶样本的检测范围为102～106cfu/μl。从细菌核酸提取至完成检测约需3h。[结论]基于Taqman探针技术的实时PCR检测体系可同步检测O157大肠杆菌rfbE和stx2基因,具有快速、灵敏度高、特异性强等优点,可用于O157大肠杆菌食物中毒的快速诊断和食品微生物检测。

外文摘要：[Objective] To establish a duplex real-time PCR assay to detect rfbE gene and stx2 gene rapidly and synchronously. [Methods] Two combinations of primers and Taqman-based fluorescent probes were designed according to the sequences of rfbE gene and stx2 genc published by GenBank, and the reaction condition were optimized to set up a duplex Taqman-based real-time PCR assay. Simultaneously, the specificity, sensitivity and repetitiveness of the assay were assessed. [Results] The real-time PCR assay successfully distinguished the E.coli O157 serotype front non-E.coli O157 serotypes and other common enteric bacterial strains. All bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assays were linear for DNA concentrations of E.coli O157 in pure culture, ranging from 10^2 to 10^6 cfu/μl and the detection limit of the real-time PCR assay ranged from 10^2 to 10^6 cfu/μl for contaminated milk samples. The overall test could be finished within about 3 hours. [Conclusions] The duplex Taqman-based real-time PCR assay for rfbE gene and stx2 gene are proved to be a rapid, sensitive and specific test for the quantitative detection of E.coli O157 from the contaminated food samples.