McMahon Tanis, Clarke Sarah, Deschênes Mylène, Tapp Kyle, Blais Burton, Gill Alexander
Health Canada/Santé Canada, Bureau of Microbial Hazards, Ottawa, Ontario, Canada.
Canadian Food Inspection Agency/Agence canadienne d'inspection des aliments, Ottawa Laboratory Carling, Ottawa, Ontario, Canada.
Int J Food Microbiol. 2024 Jul 16;419:110744. doi: 10.1016/j.ijfoodmicro.2024.110744. Epub 2024 May 11.
Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes. A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence. The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.
产志贺毒素大肠杆菌（STEC）是食源性肠道病原体。通过检测志贺毒素（Stx）或其基因（stx）可将STEC与其他大肠杆菌区分开来。已确定的Stx命名法识别出十种亚型（Stx1a、Stx1c、Stxd、Stx2a至Stx2g）。另外还报告并描述了九种亚型（Stx1e、Stx2h至Stx2o）。许多聚合酶链反应（PCR）方案仅能检测部分Stx亚型，这限制了它们的包容性。在此，我们描述了一种包含所有当前描述的Stx亚型代表DNA序列的实时PCR检测方法。使用九条引物和四条探针开发了一种用于检测stx的多重实时PCR检测方法。由于STEC的鉴定不需要区分stx亚型，因此这些探针使用相同的荧光报告基团，以便在单个反应中检测多个可能的靶标。PCR混合物包含一个内部阳性对照，以检测反应抑制情况。因此，该方案可在双通道实时PCR平台上进行。为降低使用STEC培养物作为过程对照所固有的生物安全风险，该方案还包括使用携带编码stx2a序列靶向片段质粒的非致病性大肠杆菌转化体的选项。针对137株STEC菌株和一株痢疾志贺氏菌的菌落评估了PCR的包容性，包括携带代表十四种亚型（stx1a、c、d；stx2a - j和o）的单拷贝stx的菌株。另外五种亚型（stx1e、2k、2l、2m和2n）由携带编码类毒素（酶无活性A亚基）序列质粒的大肠杆菌转化体代表。特异性检测组由70种细菌组成，包括21种stx阴性大肠杆菌。用人工接种的碎牛肉、菠菜、奶酪和苹果酒评估了该方法对食品分析的适用性。实时PCR对由STEC、痢疾志贺氏菌和携带stx类毒素质粒的大肠杆菌转化体菌落代表的所有19种stx亚型均产生了阳性结果。特异性检测组菌落的检测均为阴性。实时PCR检测到所有测试的接种食品富集物中均存在stx，并且通过分离确认了STEC的存在。
