Departamento de Patologia Médica, Universidade Federal do Paraná, Curitiba-PR, Brazil.
J Clin Lab Anal. 2013 Mar;27(2):155-61. doi: 10.1002/jcla.21578. Epub 2013 Feb 19.
Diarrheagenic Escherichia coli (DEC) strains are important causes of diarrhea. However, they cannot be distinguished from E. coli of the intestinal microbiota by conventional microbiological tests.
This work presents a two-system multiplex PCR for detection of DEC. Primers for 16S rRNA gene were added as internal amplification control to validate negative reactions. The multiplex-PCR system 1 contains primers for detection of Shiga toxin producing E. coli (STEC; stx1, stx2), enteropathogenic E. coli (EPEC; eae, bfpA), atypical enteropathogenic E. coli (aEPEc; eae), enteroinvasive E. coli (ETEC; lt, st), enteroinvasive E. coli (EIEC; ial), and the internal amplification control 16S rRNA. The system 2 contains primers for EIEC (ipaH), enteroaggregative E. coli (CVD432), diffusely adherent E. coli (daaE), and 16S rRNA. The protocol was tested with E. coli reference strains, and also with cultures of fecal specimens of people with diarrhea and healthy controls.
The protocol correctly identified the DEC reference strains. No DEC marker was amplified for negative controls; these results were validated by the amplification of a fragment of the 16S rRNA gene. The frequency of DEC was 7.6% for both patients and healthy controls; two Shigella sonnei strains were detected in the group with diarrhea. The identity of the amplicons was confirmed by DNA sequencing.
The protocol is specific for DEC Shigella and is suitable for clinical laboratories.
产肠毒性大肠杆菌（DEC）菌株是腹泻的重要病因。然而，它们不能通过常规微生物学检测与肠道微生物群中的大肠杆菌区分开来。
本研究提出了一种用于检测 DEC 的双系统多重 PCR 方法。为 16S rRNA 基因添加引物作为内部扩增对照，以验证阴性反应。该多重 PCR 系统 1 包含用于检测产志贺毒素大肠杆菌（STEC；stx1、stx2）、肠致病性大肠杆菌（EPEC；eae、bfpA）、非典型肠致病性大肠杆菌（aEPEC；eae）、肠侵袭性大肠杆菌（ETEC；lt、st）、肠侵袭性大肠杆菌（EIEC；ial）和内部扩增对照 16S rRNA 的引物。系统 2 包含用于 EIEC（ipaH）、肠聚集性大肠杆菌（CVD432）、弥散粘附性大肠杆菌（daaE）和 16S rRNA 的引物。该方案用大肠杆菌参考菌株进行了测试，也用腹泻患者和健康对照者的粪便标本培养物进行了测试。
该方案正确鉴定了 DEC 参考菌株。阴性对照未扩增 DEC 标志物；这些结果通过扩增 16S rRNA 基因的片段得到验证。患者和健康对照者的 DEC 检出率均为 7.6%；在腹泻组中检测到两株宋内志贺菌。扩增子的同一性通过 DNA 测序得到确认。
该方案针对 DEC 志贺菌具有特异性，适合临床实验室使用。
