Development of PCR-based ORF typing method for multiple serotypes of Shiga toxin- producing <i>Escherichia coli</i>.

Escherichia coli O157 and O26 are well-known Shiga toxin-producing E. coli (STEC) serotypes, but other serotypes such as O91, O103, O111, O121, O145, and O165 have also emerged as important public health concerns. PCR-based methods, such as multilocus variable-number tandem-repeat analysis (MLVA), are commonly used for rapid genotyping of STEC strains. However, MLVA requires analysis of numerous loci, some of which are serotype-specific, limiting its applicability across multiple STEC serotypes. In this study, we developed a PCR-based open reading frame typing (POT) method for genotyping diverse STEC serotypes. By detecting 22 ORFs using two multiplex PCR assays, STEC genotypes were determined based on the presence or absence of specific ORFs. Most STEC serotypes, except for O121, were classified with high discriminatory power (Simpson's index: 0.95-0.98). STEC-POT represents a rapid, cost-effective, and broadly applicable molecular typing tool. It facilitates timely outbreak detection and enhances public health surveillance by enabling rapid molecular epidemiological investigations.