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 re action (PCR) protocol was de eloped for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O 157 Escherichia coli isolates. Using shiga toxin gene-specific primers, com bined with two short 10-mer primers, in a multiples shiga toxin/RAPD-PCR th e fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the s higa toxin gene(s). Hybridization with a digoxigenin-labelled probe specifi c for stx1 and stx2 confirmed its identity. The combination of primers in t his way allows valuable additional information to be gained from discrimina tory RAPD profiles, with further benefits of time and cost savings over tes ts performed individually.