A Simple PCR-Based Macroarray System for Detection of Multiple Gene Markers in the Identification of Priority Enterohemorrhagic Escherichia coli
A Simple PCR-Based Macroarray System for Detection of Multiple Gene Markers in the Identification of Priority Enterohemorrhagic Escherichia coli
复制标题
DOI：
10.4315/0362-028x.jfp-10-450
复制
发表时间：
2011-03-01
影响因子：
2
通讯作者：
Martinez-Perez, Amalia
中科院分区：
文献类型：
--
作者：
Blais, Burton W.;Martinez-Perez, Amalia
携带O抗原决定簇O 157、O26、O 111、O 103和O 145的肠出血性大肠杆菌（EHEC）菌株在全球范围内与食源性疾病的关联率很高。为了扩大加拿大的食品检查能力，开发了一种基于布的杂交阵列系统（CHAS），用于优先鉴定和表征肠出血性大肠杆菌。该方法针对关键毒力基因（eae、hlyA、vt 1和vt 2）加上指定O 157抗原决定簇的rfbE基因，以及指定O26、O 111、O 103和O 145决定簇的方式基因。通过与固定在聚酯布支持物上的特异性寡核苷酸探针阵列杂交，检测掺入地高辛标记的多重PCR产物，随后对捕获的扩增子进行免疫酶测定。该方法鉴定了85种不同菌株中的相关标记，这些菌株具有靶基因的各种组合（毒力和优先O-抗原标记）。在26株其它大肠杆菌中均未检测到目的基因。coli和non-E.大肠杆菌CHAS表现出100%的包容性和100%的排他性的特点，在不同的细菌菌株之间的各种标志物的检测。CHAS表现出100%的包容性和100%的排他性的特点，在各种目标和非目标细菌之间的标志物的检测。整个过程可以在不到5 h内完成，并且可用于通过使用富集培养技术鉴定从食品中分离的优先EHEC菌落。
Enterohemorrhagic Escherichia coli (EHEC) strains bearing the O antigenic determinants O157, O26, O111, O103, and O145 have a high rate of association with foodborne illness worldwide. To expand Canadian food inspection capability, a cloth-based hybridization array system (CHAS) was developed for the identification and characterization of priority EHEC. This method targets key virulence genes (eae, hlyA, vt1, and vt2) plus the rfbE gene specifying the O157 antigenic determinant, and the way genes specifying the O26, O111, O103, and O145 determinants. Multiplex PCR products incorporating a digoxigenin label were detected by hybridization with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. This method identified the relevant markers in 85 different strains bearing various combinations of the target genes (virulence and priority O-antigen markers). None of the target genes was detected in 26 different strains of other E. coli and non-E. coli bacteria. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the various markers among different bacterial strains. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the markers among various target and nontarget bacteria. The entire procedure could be completed in less than 5 h, and is useful for the identification of priority EHEC colonies isolated from foods by using enrichment culture techniques.