Abstract: Aims: To develop a duplex real-time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2-encoding genes which are pivotal to EHEC-mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non- E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H -, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H -, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real-time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19.75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real-time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real-time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.