State Key Laboratory for Infectious Disease Prevention and Control. National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Changping District, Beijing, 102206, China.
Shanghai Changning District Center for Disease Control and Prevention, 39 Yunwushan Road, Changning District, Shanghai, 200051, China.
Mol Cell Probes. 2020 Feb;49:101477. doi: 10.1016/j.mcp.2019.101477. Epub 2019 Nov 1.
Diarrheagenic Escherichia coli (DEC) is a set of the most common pathogens causing diarrhea. DEC strains are classified into five pathotypes based on the possession of different virulence genes: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) or Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). The development of an easy-to-use method to detect the specific virulence genes and distinguish the pathotypes is essential for the diagnosis and surveillance of DEC infections. In this study, a multiplex PCR assay (mPCR) specific to nine virulence genes and an internal control was designed for the identification of five DEC pathotypes. A temperature switch PCR (TSP) strategy was used in the PCR amplification. The PCR products were detected by capillary electrophoresis. The limit of detection (LOD) of the 10-plex reaction was 5 × 10 copies/reaction for stx2 and 5 × 10 copies/reaction for the other targets. The mPCR showed very high specificity, and inclusivity and exclusivity were both 100%. When the mPCR assay was used for the detection of 221 cryopreserved diarrhea specimens, DEC colonies were detected from 49 specimens, and the positive rate was 22.2%. The mPCR assay was sensitive and specific, and the amplified product could be analyzed easily. Thus, this method could be used effectively to identify the suspected colonies of DEC in the primary culture of the specimen.
产毒性大肠杆菌（DEC）是一组引起腹泻的最常见病原体。根据不同毒力基因的存在，DEC 菌株可分为五种病原型：肠致病性大肠杆菌（EPEC）、肠出血性大肠杆菌（EHEC）或产志贺毒素大肠杆菌（STEC）、肠聚集性大肠杆菌（EAEC）、肠产毒性大肠杆菌（ETEC）和肠侵袭性大肠杆菌（EIEC）。开发一种易于使用的方法来检测特定的毒力基因并区分病原型，对于 DEC 感染的诊断和监测至关重要。在本研究中，设计了一种针对九个毒力基因的多重 PCR 检测方法（mPCR）和一个内部对照，用于鉴定五种 DEC 病原型。PCR 扩增采用温度切换 PCR（TSP）策略。通过毛细管电泳检测 PCR 产物。10 重反应的检测限（LOD）为 stx2 的 5×10 拷贝/反应，其他靶标的 5×10 拷贝/反应。mPCR 显示出非常高的特异性，包容性和排他性均为 100%。当使用 mPCR 检测 221 份冷冻保存的腹泻标本时，从 49 份标本中检测到 DEC 菌，阳性率为 22.2%。mPCR 检测方法敏感且特异性高，扩增产物易于分析。因此，该方法可有效用于鉴定标本初步培养中 DEC 的疑似菌落。
