Abstract：
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.
摘要：
产志贺毒素的大肠杆菌（STEC）是食源性肠道病原体。通过检测志贺毒素(Stx)或其基因(stx)将STEC与其他大肠杆菌区分开。Stx的既定命名法标识了十个子类型(Stx1a，Stx1c,Stxd,Stx2a到Stx2g)。已经报道和描述了另外9种亚型(Stx1e，Stx2h到Stx2o)。许多PCR方案仅检测限制其包容性的Stx亚型的子集。在这里，我们描述了实时PCR测定，包括所有目前描述的Stx亚型的代表的DNA序列。使用9种引物和4种探针开发了用于检测stx的多重实时PCR测定法。由于STEC的鉴定不需要区分stx亚型，探针使用相同的荧光报告子，以便在单个反应中检测多个可能的目标。PCR混合物包括内部阳性对照以检测反应的抑制。因此，该协议可以在两通道实时PCR平台上执行。为了降低使用STEC培养物作为过程控制所固有的生物安全风险，该方案还包括选择携带编码stx2a序列的靶向片段的质粒的非致病性大肠杆菌转化体。对137株STEC菌株和一株痢疾志贺氏菌的菌落进行了PCR的包容性评估，包括携带代表14种亚型的stx单拷贝的菌株(stx1a，C,d;stx2a-j和o)。五个额外的子类型(stx1e,2k,2l,2m和2n）由编码类毒素（酶失活的A亚基）序列的质粒转化的大肠杆菌代表。排他性小组由70个细菌组成，包括21个stx阴性大肠杆菌。用人工接种的碎牛肉评估食物分析的适宜性，菠菜,奶酪,和苹果酒。实时PCR对所有19种stx亚型都产生了阳性结果，以STEC殖民地为代表，携带stx类毒素质粒的痢疾链球菌和大肠杆菌转化体。排他性小组菌落的测试都是阴性的。实时PCR检测到所有接种的食物富集物中都存在stx，并且通过分离证实了STEC的存在。
