French Agency for Food, Environmental and Occupational Health and Safety, Food Safety Laboratory, Maisons-Alfort, France.
J Clin Microbiol. 2012 Nov;50(11):3485-92. doi: 10.1128/JCM.01656-12. Epub 2012 Aug 15.
In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR(O104:H4)) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR(O104:H4) PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC(O104), wzx(O104), and wzy(O104)). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR(O104:H4) target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR(O104:H4) locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPR(O104:H4) PCR (99.06% specificity).
2011 年，欧洲发生了一次大规模的不寻常细菌菌株爆发。该菌株被鉴定为肠聚集性大肠杆菌（EAEC）和产志贺毒素大肠杆菌（STEC）O104:H4 血清型菌株的杂交体。在这里，我们提出了一种针对大肠杆菌 O104:H4 的簇状规则间隔短回文重复序列（CRISPR（O104:H4））的单一 PCR，用于从不同地理位置和时间段特异性检测 EAEC STEC O104:H4 菌株。使用 1321 株大肠杆菌菌株，包括大肠杆菌 O 血清群 O1 至 O186 和鞭毛（H）型 H1 至 H56 的参考菌株，研究了 CRISPR（O104:H4）PCR 的特异性。使用针对不同 O104 抗原编码基因（wbwC（O104）、wzx（O104）和 wzy（O104））的 PCR 检测方法比较了该检测方法的特异性。PCR 检测方法与所有类型的大肠杆菌 O104 菌株（O104：H2、O104：H4、O104：H7 和 O104：H21）和携带 K9 荚膜抗原的大肠杆菌 O8 和 O9 菌株反应，因此不能特异性检测 EAEC STEC O104：H4 型。针对 CRISPR（O104:H4）靶标开发的单个 PCR 足以特异性鉴定和检测 48 株测试的 EAEC STEC O104：H4 菌株。表达 H 型除 H4 以外的 35 株大肠杆菌 O104 菌株以及携带 K9 荚膜抗原的 8 株大肠杆菌菌株在 CRISPR（O104：H4）基因座上均为阴性。在 1273 株非 O104：H4 大肠杆菌菌株（血清型 Ont：H2、O43：H2、O141：H2 和 O174：H2）中，只有 12 株（0.94%）在 CRISPR（O104：H4）PCR 中呈阳性反应（特异性 99.06%）。
