PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as <10 CFU/ml. The O26-and O113-specific PCR assays can potentially be used for typing E. coli O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.Escherichia coli O26 strains, first isolated from cases of infantile diarrhea, have been implicated in causing hemolytic uremic syndrome (HUS) (13) and serious enteric disorders in humans in the United Kingdom (12), Germany (17), Poland (9), Spain (2), and Finland (7). Among the non-O157 Shiga toxin-producing E. coli (STEC) isolates, O26 has been the most common serogroup, composing 18% (1,066 of 5,913) of the total number of STEC isolates reported from 1997 to 1999 (6). E. coli O26 strains have been found to be genetically diverse with unique virulence profiles (19). An eae-negative O113:H21 STEC strain was responsible for an HUS outbreak in South Australia (10). Since traditional E. coli growth and isolation methods show all non-O157 STEC to be phenotypically similar to nonpathogenic E. coli, detection of specific STEC serogroups is problematic. There is no rapid method presently available for detecting specific STEC strains.The O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria and consists of many repeats of an oligosaccharide unit (O unit). The O antigen is the major contributor of antigenic variability on the cell surface, and on this basis different O types have been designated. The genes involved in the biosynthesis of O antigens in E. coli are generally clustered and flanked by the galF and gnd genes at the 5Ј and 3Ј ends, respectively. O-antigen gene clusters including O26 and O113 have been cloned and sequenced (4, 11). Analyses of each of the genes in the cluster by National Center for Biotechnology Information genome BLAST and gene alignment software programs showed that the O-unit flippase gene (wzx) and the polymerase gene (wzy) were unique for E. coli O26 as well as for O113 antigens. Therefore, these genes were targeted for developing PCR assays for detecting these serogroups.All E. coli strains used in the study were from the bacterial collection of the Gastroenteric Disease Center at The Pennsylvania State University. Reference standard strains, E. coli O26:HϪ (H31b) and E. coli O113:H21 (6182-50) from the World Health Organization (8), were used for developing the assays. The 179 World Health Organization O reference ...