This Technical Specification describes the identification of Shiga toxin-producing Escherichia coli (STEC) by means of the detection of the following genes: a) the major virulence genes of STEC, stx and eae (References ); b) the genes associated with the serogroups O157, O111, O26, O103, and O145 (References ). In any case, when one or both of the stx genes is/are detected, the isolation of the strain is attempted. The isolation of STEC from samples positive for the presence of the genes specifying the serogroups in the scope of this method can be facilitated by using serogroup-specific enrichment techniques (e.g. Immunomagnetic separation, IMS). The protocol uses real-time PCR as the reference technology for detection of the virulence and serogroupassociated genes. This Technical Specification is applicable to: 1) products intended for human consumption and the feeding of animals; 2) environmental samples in the area of food production and food handling; 3) environmental samples in the area of primary production.