Single multiplex PCR assay to identify the shiga toxin
Strains of shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae type1 have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. Most clinical signs of disease arise as consequence of the production of shiga toxin/shiga toxin 1 (Stx/Stx1), shiga toxin 2 (Stx2) or combination of these toxins. Here, we designed a Multiplex PCR technique to identify stx/stx1 and stx2 genes with the incorporation of mdh gene of E. coli and Shigella. A total of six primers were used: SFI and SRI, which produce a 199bp product that serves as an internal positive control; Ka2F and Ka2R, which yield a 381bp fragment of stx2 gene, and Ka1F and Ka1R, which amplify a 622bp fragment of stx/stx1. The thermal profile, which was preceded by a 5 min incubation at 95 degrees C for 20 to 25 cycles with the following parameters: 95 degrees C at 1 min, 60 degrees C at 1 min, 72 degrees C at 1 min, and 5 min incubation at 72 degrees C as final extension. PCR amplification products identifying the stx/stx1 and stx2 gene sequences were observed only in E. coli 0157:H7 and Shigella dysenteriae type1. Template nucleic acid extracted from other Gram-negative bacteria was found to be negative. The sensitivity of the PCR procedure for detection of shiga toxin genes was determined to be 2.1 pg/mu l of total nucleic acid and 320 cfu/mu l.