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 developed 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 multiplex 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.
Hopkins
K L
KL
School of Biosciences and Division of Environmental Health and Risk Management, University of Birmingham, Edgbaston, Birmingham, UK.
Hilton
A C
AC
D001427:Bacterial Toxins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D015139:Blotting, Southern; D004926:Escherichia coli / Q000145:classification* / Q000235:genetics*; D004927:Escherichia coli Infections / Q000453:epidemiology / Q000382:microbiology; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D019105:Random Amplified Polymorphic DNA Technique; D022603:Shiga Toxins
D001427:Bacterial Toxins; D022603:Shiga Toxins