A rapid method for the discrimination of genes encoding classical Shiga toxin (Stx) 1 and its variants, Stx1c and Stx1d, in Escherichia coli

Subtyping of Shiga toxin (Stx)‐encoding genes by conventional polymerase chain reaction (PCR) is time‐consuming. We developed a single step real‐time fluorescence PCR with melting curve analysis to distinguish rapidly stx 1 from its variants, stx 1c and stx 1d . Melting temperatures ( T m ) of 206 Stx‐producing Escherichia coli (STEC) identified to harbor stx 1 or stx 1c were analyzed using a specific hybridization probe over the variable region. 170 of 171 stx 1 ‐harboring STEC displayed T m of 69°C to 70°C, whereas 34 of 35 strains containing stx 1c had T m of 65°C–66°C. This constant and reproducible difference of 4°C demonstrated that melting curve analysis is a reliable technique to differentiate stx 1 from stx 1c . Two isolates displayed atypical T m . Sequence analysis showed that one of them was 100% identical to stx 1d within a 511 bp DNA stretch. Our data demonstrate that real‐time PCR is a rapid and reliable tool to differentiate stx 1 from stx 1c and stx 1d and to detect new stx 1 variants. Because stx 1 ‐harboring STEC cause diarrhoea and hemolytic‐uremic syndrome, whereas those containing stx 1c are often shed asymptomatically, a rapid differentiation between stx 1 and its variants using the procedure developed here has both clinical implications and a direct significance for the risk assessment analysis of STEC isolated from foods.