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 × 103 copies/reaction for stx2 and 5 × 102 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.

中文翻译：

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通过新的多重PCR分析和毛细管电泳鉴定腹泻性大肠杆菌。

腹泻性大肠杆菌（DEC）是一组引起腹泻的最常见病原体。根据拥有不同毒力基因，DEC菌株可分为五种致病型：肠致病性大肠杆菌（EPEC），肠出血性大肠杆菌（EHEC）或产生志贺毒素的大肠杆菌（STEC），肠聚集性大肠杆菌（EAEC） ，产肠毒素的大肠杆菌（ETEC）和肠侵害性大肠杆菌（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体对于DEC感染的诊断和监视至关重要。在这项研究中，针对9个毒力基因和一个内部对照的多重PCR检测（mPCR）设计用于鉴定5种DEC致病型。在PCR扩增中使用温度开关PCR（TSP）策略。通过毛细管电泳检测PCR产物。10重反应的检测下限（std2）为5×103拷贝/反应，其他靶标为5×102拷贝/反应。mPCR显示非常高的特异性，包容性和排他性均为100％。用mPCR检测221份冷冻保存的腹泻标本时，从49份标本中检测出DEC菌落，阳性率为22.2％。mPCR检测灵敏且特异，扩增产物易于分析。因此，该方法可有效用于鉴定标本原代培养物中可疑的DEC菌落。mPCR显示非常高的特异性，包容性和排他性均为100％。用mPCR检测221份冷冻保存的腹泻标本时，从49份标本中检测出DEC菌落，阳性率为22.2％。mPCR检测灵敏且特异，扩增产物易于分析。因此，该方法可有效地用于鉴定样本原代培养物中可疑的DEC菌落。mPCR显示非常高的特异性，包容性和排他性均为100％。mPCR法检测221份冷冻保存的腹泻标本，从49份标本中检测出DEC菌落，阳性率为22.2％。mPCR检测灵敏且特异，扩增产物易于分析。因此，该方法可有效地用于鉴定样本原代培养物中可疑的DEC菌落。