Pattabiraman Vaishnavi, Parsons Michele B, Bopp Cheryl A
National Center for Zoonotic and Emerging Infectious Diseases, Centers for Disease Control and Prevention , Atlanta, Georgia .
Foodborne Pathog Dis. 2016 Apr;13(4):212-20. doi: 10.1089/fpd.2015.2064. Epub 2016 Feb 9.
Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea in children under the age of 5 years in developing countries and are the leading bacterial agent of traveler's diarrhea in persons traveling to these countries. ETEC strains secrete heat-labile (LT) and/or heat-stable (ST) enterotoxins that induce diarrhea by causing water and electrolyte imbalance. We describe the validation of a real-time TaqMan PCR (RT-PCR) assay to detect LT, ST1a, and ST1b enterotoxin genes in E. coli strains and in stool specimens. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay using a conventional PCR assay as a gold standard with 188 ETEC strains and 42 non-ETEC strains. We validated LT/ST1b duplex and ST1a single-plex RT-PCR assay in stool specimens (n = 106) using traditional culture as the gold standard. RT- PCR assay sensitivities for LT, ST1a, and ST1b detection in strains were 100%, 100%, and 98%; specificities were 95%, 98%, and 99%, and Pearson correlation coefficient r was 0.9954 between RT-PCR assay and the gold standard. In stool specimens, RT-PCR assay sensitivities for LT, ST1a, and ST1b detection were 97%, 100%, and 97%; and specificities were 99%, 94%, and 97%. Pearson correlation coefficient r was 0.9975 between RT-PCR results in stool specimens and the gold standard. Limits of detection of LT, ST1a, and ST1b by RT-PCR assay were 0.1 to1.0 pg/μL and by conventional PCR assay were 100 to1000 pg/μL. The accuracy, rapidity and sensitivity of this RT-PCR assay is promising for ETEC detection in public health/clinical laboratories and for laboratories in need of an independent method to confirm results of other culture independent diagnostic tests.
产肠毒素大肠杆菌（ETEC）是发展中国家5岁以下儿童腹泻的重要病因，也是前往这些国家的旅行者腹泻的主要细菌病原体。ETEC菌株分泌热不稳定（LT）和/或热稳定（ST）肠毒素，通过引起水和电解质失衡导致腹泻。我们描述了一种实时TaqMan PCR（RT-PCR）检测方法的验证，该方法用于检测大肠杆菌菌株和粪便标本中的LT、ST1a和ST1b肠毒素基因。我们使用传统PCR检测作为金标准，对188株ETEC菌株和42株非ETEC菌株验证了LT/ST1b双重和ST1a单重RT-PCR检测方法。我们使用传统培养作为金标准，在粪便标本（n = 106）中验证了LT/ST1b双重和ST1a单重RT-PCR检测方法。RT-PCR检测方法对菌株中LT、ST1a和ST1b检测的灵敏度分别为100%、100%和98%；特异性分别为95%、98%和99%，RT-PCR检测方法与金标准之间的Pearson相关系数r为0.9954。在粪便标本中，RT-PCR检测方法对LT、ST1a和ST1b检测的灵敏度分别为97%、100%和97%；特异性分别为99%、94%和97%。粪便标本中RT-PCR结果与金标准之间的Pearson相关系数r为0.9975。RT-PCR检测方法对LT、ST1a和ST1b的检测限为0.1至1.0 pg/μL，传统PCR检测方法的检测限为100至1000 pg/μL。这种RT-PCR检测方法的准确性、快速性和灵敏度对于公共卫生/临床实验室以及需要独立方法来确认其他非培养诊断测试结果的实验室进行ETEC检测很有前景。
