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) frorn 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 69degreesC to 70degreesC. whereas 34 of 35 strains containing stx(1c) had T-m of 65degreesC-66degreesC. 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 sty, 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.