Rapid and Reliable Detection of Shiga Toxin-Producing Escherichia coli by Real-Time Multiplex PCR
Rapid and Reliable Detection of Shiga Toxin-Producing Escherichia coli by Real-Time Multiplex PCR
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DOI：
10.4315/0362-028x.jfp-11-392
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发表时间：
2012-04-01
影响因子：
2
通讯作者：
Doepfer, Doerte
中科院分区：
文献类型：
--
作者：
Anklam, Kelly S.;Kanankege, Kaushi S. T.;Doepfer, Doerte
大肠杆菌O26、O 45、O 103、O 111、O 121、O 145和O 157是主要产滋贺毒素的大肠杆菌。大肠杆菌（STEC）血清群与全球人类食源性疾病爆发有关。这些病原体的日益流行具有重要的公共卫生意义。牛肉制品一直被认为是食源性人类STEC感染的主要来源。需要稳健和敏感的方法来检测和表征这些病原体，以确定牛肉加工设施中STEC的患病率和发病率，并改善旨在消除食品供应中STEC的食品安全干预措施。本研究旨在开发Taqman实时多重PCR检测方法，用于筛查和快速检测与人类疾病相关的主要STEC血清群。三种血清群特异性检测针对E.大肠杆菌O26（wzy）、O 103（wzx）和O 145（wzx），测定2中的O 45（wry）、O 111（manC）和O 121（way），以及测定3中的O 157（rfbE）。uidA基因也作为E. coli内部扩增对照。开发了第四种测定法以靶向滋贺毒素（stx（1）和stx（2））、紧密粘附素（eae）和肠溶血素（ehxA）的选定毒力基因。通过检测100株和62株E.大肠杆菌菌株和非大肠杆菌菌株。coli对照菌株。该检测试剂正确检测了所有受检菌株中的基因，未观察到交叉反应，代表100%特异性。对于纯培养物和人工污染的粪便样品，测定的检测限为10（3）或10（4）CFU/ml，在6 h富集期后，测定的检测限为10（0）CFU/ml。这些结果表明，四种实时多重PCR检测方法是稳健和有效的快速和可靠的检测主要公共卫生关注的7个主要STEC血清群及其毒力基因的检测。
Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wry), O111 (manC), and O121 (way) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100% specificity. The detection limits of the assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.