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

Abstract

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 stx1 from its variants, stx1c and stx1d. Melting temperatures (Tm) of 206 Stx-producing Escherichia coli (STEC) identified to harbor stx1 or stx1c were analyzed using a specific hybridization probe over the variable region. 170 of 171 stx1-harboring STEC displayed Tm of 69 degrees C to 70 degrees C, whereas 34 of 35 strains containing stx1c had Tm of 65 degrees C-66 degrees C. This constant and reproducible difference of 4 degrees C demonstrated that melting curve analysis is a reliable technique to differentiate stx1 from stx1c. Two isolates displayed atypical Tm. Sequence analysis showed that one of them was 100% identical to stx1d within a 511 bp DNA stretch. Our data demonstrate that real-time PCR is a rapid and reliable tool to differentiate stx1 from stx1c and stx1d and to detect new stx1 variants. Because stx1-harboring STEC cause diarrhoea and hemolytic-uremic syndrome, whereas those containing stx1c are often shed asymptomatically, a rapid differentiation between stx1 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.