双重实时荧光PCR快速检测O157大肠杆菌的初步研究  

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] 

机构地区：[1]绍兴文理学院医学院,绍兴312000 [2]浙江省疾病预防控制中心

出　　处：《现代预防医学》2007年第14期2611-2613,共3页Modern Preventive Medicine

基　　金：浙江省卫生厅医药卫生科学研究基金资助(2005A102)

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。[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.

关 键 词：实时PCR TAQMAN探针 O157大肠杆菌 检测 

分 类 号：R378.2[医药卫生—病原生物学]