Specific Identification of Escherichia coli O157:H7 Using a Multiplex PCR Assay

Abstract

Entero-hemorrhagic Escherichia coli (EHEC) are associated with hemorrhagic colitis and the hemolytic uremic syndrome in humans. EHEC produce one or more antigenic type of Vero toxin (VT) and possess a gene designated eae, which is associated with attachment to- and effacement of the microvilli of enterocytes. E. coli O157:H7 is the most common EHEC serotype isolated and this organism has been associated with a number of serious outbreaks of food-borne disease. Cultural methods for identification of E. coli O157:H7 rely on selective media such as Cefixime Tellurite Sorbitol MacConkey Agar (Chapman et al., 1993), serotyping and toxin testing. This approach is labor intensive and may take a week or more to complete. In addition, certain other bacteria do not ferment sorbitol, grow on this selective media and cross-react in slide agglutination reactions with antiserum to the O157 lipopolysaccharide. Sorbitol-fermenting E. coli O157 belonging to the EHEC group have also been reported (Karch et al., 1993). We have previously described methods which allow rapid identification of E. coli O157:H7 and other EHEC using polymerase chain reaction (PCR) assays which target vt and eae genes (Gannon et al., 1992, Gannon et al., 1993). In these studies, so called “generic” EHEC PCR primer sets were developed which target vt genes and a 5′ conserved area of the eae gene. These PCR primers allow identification of all EHEC. In addition, a PCR assay was devised which is highly specific for E. coli O157:H7. This PCR assay uses the 3′ end of the eae gene of E. coli O157:H7 (eae _O157) as a target for amplification. Unfortunately, strains of the enteropathogenic E. coli (EPEC) serotype O55:H7 and the EHEC serotype O145:H- are also positive in PCR assays with eae _O157 oligonucleotide primers.