Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current Methods: of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fl uorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to speci fi cally amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli (EAEC), stIa/ stIb and lt for enterotoxigenic E. coli (ETEC), eaeA for enteropathogenic E. coli (EPEC), stx1 and stx2 for Shiga toxin-producing E. coli (STEC), ipaH for enteroinvasive E. coli (EIEC), and daaD for diffusely adherent E. coli (DAEC).