Abstract: Objective: To develop a multiplex real-time PCR method for rapid detection and identification of five strains of diarrheagenic Escherichia coli. Methods: The multiplex real-time PCR method was established based on 11 virulence genes, including eae, stx1, stx2, ipaH, uidA, estla, estlb, aggR, pic, elt and astA. This method consisted of two reaction systems, A and B, which could be used to detect diarrheagenic enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC), respectively. The concentrations of probe and primers and PCR reaction conditions were optimized, and the sensitivity and specificity of the method were finally tested. Results: The primers and probes specifically amplified 11 gene fragments, and the sensitivities of systems A and B were 2.6 × 104 and 2.2 × 104 copies/reaction, respectively. Conclusion: The multiplex real-time PCR method established in this study can be used to determine whether one sample is diarrheagenic E. coli and which type of pathogen it belongs to by the simultaneous use of reactions A and B. This method can provide a basis for clinical detection, disease prevention and outbreak traceability of diarrheagenic E. coli.

Key words: diarrheagenic Escherichia coli, multiplex real-time PCR, virulence gene