Strains of shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae type 1 have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in human. Most clinical signs of disease arise as a consequence of the production of shiga toxin! shiga toxin 1 (Stx! Stxl), shiga toxin2 (Stx2) or combination of these toxins. The aim of this study was detection of shiga toxin genes by using molecular technic.
A technique has been developed for detection of stx! stxl and stx2 genes by using the multiplex polymerase chain reaction assay with incorporation of mdh gene of E. coli and Shigella. A total of six primers were used: SF1 and SRI, which produce a 199 bp 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 622 bp fragment of stx! stxl. Restriction enzyme-digestion and sequencing were used for confirmation of these fragments.
PCR amplification products identifing the stx!stxl and stx2 gene sequences were observed only in E. coli 0157:H7 and shigella dysenteriae typel. Template nucleic acid extracted from other gram-negative bacteria was found to be negative. These fragments were confirmed by using restriction enzyme-digestion and sequencing. The sensitivity of the PCR procedure for detection of shiga toxin genes was determined to be 2.1 pg!.il of total nucleic acid and 320 cfu!jil.
A multiplex PCR which detects genes encoding Mdh, Stx!Stxl and Stx2 has been developed. E. coli 0157:H7 produces Stxl and Stx2, whereas Shigella dysenteriae biotype 1 produces only Stx. Using this research and comparing with genome bank, we found that the fragment belonging to stx2 is suitable for detection of all kinds of stx2 genes. This technique is a useful diagnostic tool for detection of shiga toxins because it is quick, specific, sensitive, and relatively inexpensive.