Development of a Rapid PCR Method Using the Insertion Sequence IS 1203 for Genotyping Shiga Toxin-Producing Escherichia coli O157

Abstract

ABSTRACT We developed a rapid PCR method utilizing the diversity of the insertion site IS 1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS 1203 PCR typing). DNA fragments digested by PvuII, which cut IS 1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS 1203 . To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS 1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS 1203 PCR typing ( D = 0.974) is similar to that of PFGE ( D = 0.981). This method can be used for rapid and simplified genotyping.