Evaluation of a PCR-restriction fragment length polymorphism (PCR-RFLP)assay for molecular epidemiological study of Shiga toxin-producing Escherichia coli

In this study, we have evaluated our recently developed polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) assay for the molecular subtyping of Shiga toxin-producing Escherichia coli (STEC). A total of 200 STEC strains includingO157 (n=100), O26 (n=50), O111 (n=10), and non-O26/O111/O157 (n=40) serogroups isolated during 2005-2006 in Japan, whichwere identified to be clonally different by pulsed-field gel electrophoresis (PFGE) were further analyzed by the PCR-RFLP assay in comparisonto PFGE. Ninety-five of O157, 48 of O26, five of O111 and 19 of non-O26/O111/O157 STEC strains yielded one to three ampliconsranging from 6.0 to 15.5 kb in size by the specific primer set targeting region V which is located in the upstream of stx genes.These strains were classified into 41 (O157), 8 (O26), 4 (O111) and 17 (non-O26/O111/O157) groups based on the RFLP patternsobtained by subsequent restriction digestion, respectively. Although the discriminatory power of PCR-RFLP assay was somewhat lessthan that of PFGE, it is more convenient for molecular subtyping of STEC strains especially for O157, the most important serogroupimplicated in human diseases, as well as to identify the outbreak-associated isolates because of its simplicity, rapidity, ease and goodreproducibility.