Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction

A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was de eloped for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiples shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually.