文献类型：期刊文献

中文题名：实时PCR在大肠杆菌O157∶H7快速检测中的应用

英文题名：Rapid detection of Escherichia coli O157∶H7 by using real-time fluorescence PCR assays

作者：张建华[1];陆群英[2];程苏云[2];卢亦愚[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：23

期号：8

起止页码：839

中文期刊名：中国人兽共患病学报

外文期刊名：Chinese Journal of Zoonoses

收录：CSTPCD、、北大核心2004、CSCD2011_2012、北大核心、CSCD

基金：浙江省卫生厅医药卫生科学研究基金资助(No.2005A102)

语种：中文

中文关键词：实时PCR;Taqman探针;大肠杆菌O157:H7;检测

外文关键词：real-time fluorescence PCR; Enterohemorrhagic E. coli O157： H7; TaqMan-based orobe; detection

中文摘要：目的建立一种快速、敏感、特异的大肠杆菌O157∶H7的定量检测方法。方法根据GenBank公布的O157∶H7大肠杆菌rfbE基因序列,应用分子生物学软件进行序列比对,在该基因的保守区设计引物和TaqMan探针,对荧光定量PCR反应条件进行优化,建立实时荧光定量PCR检测大肠杆菌O157∶H7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。结果所有大肠杆菌O157∶H7菌株的检测结果均为阳性,而所有其它菌株检测结果均为阴性;该方法检测的灵敏度可达1cfu/μL,重复性检测的变异系数均小于5%。在模拟污染牛奶中,可检出1×102cfu/μL的细菌。从细菌核酸提取至完成检测约需3h。结论本研究建立的基于Taqman探针技术的实时PCR检测体系具有快速、灵敏度高、特异性强等优点,可用于大肠杆菌O157∶H7食物中毒的快速诊断和食品微生物检测,为食源性疾病的分子流行病学调查提供新的检测手段。

外文摘要：In order to establish a TaqMan-based real-time PCR assay for the detection of E. coli O157 ： HT, the primers and TaqMan-based probe were designed according to the conserved rcgion of rfbE. coli O157 gene published on GenBank. The primers, probes and the reactive condition were optimized to improve the sensitivity and specificity of the assay. The pure culture and raw milk samples were detected to evaluate the sensitivity of the assay. Analysis of 32 bacterial strains demonstrated that the real-time PCR assay successfully distinguished the E. coli O157 serotype from non-E, coli O157 scrotypes and other common enteric bacteria. All bacterial strains that lacked this gene were not detected by this assay. The quantitative ranges of the real-time PCR assay were linear for DNA concentrations ranging from 10^o to 10^4 cfu/μL of E. coli O157 in pure culture, but the detection sensitivity of the real-time PCR assay ranged from 10^2 to 10^4 cfu/μL for raw milk samples without enrichment. The overall test could be finished within about 3 hours. The TaqMan-based real-time PCR assay for rfb E. coli O157 gene was proved to be a rapid, sensitive and specific test for the quantitative detection of E. coli O157 from the contaminated food sampies.