A MULTIPLEX PCR FOR RAPID IDENTIFICATION OF SHIGA-LIKE TOXIN-PRODUCING ESCHERICHIA-COLI O157-H7 ISOLATED FROM FOODS

For rapid and specific identification of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food samples, experimental conditions for a multiplex polymerase chain reaction (PCR) were optim ized and a multiple digoxigenin (DIG)-labeled oligonucleotide probe hy bridization (DLOPH) assay was developed. A suspect colony from MacConk ey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-beta-D-glucuron ide (MSA-BCIG) was used for the multiplex PCR. Three different DNA seq uences off. coli O157:H7 were amplified simultaneously in the PCR: a s pecific fragment of an attaching and effacing gene (eae gene), conserv ed sequences of Shiga-like toxins (SLT) I and II, and a fragment of th e 60-MDa plasmid. The identities of PCR products were confirmed by hyb ridization using DIG-labeled internal oligonucleotide probes and color imetric detection with anti-DIG Fab fragments conjugated to alkaline p hosphatase. This method yielded positive results with all reference st rains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, an d O157:H-, and negative results were obtained with all strains of nont oxigenic E. coli serogroup O157, other serotypes of E. coli, and other bacterial species. The detection limit of the method was 65 colony-fo rming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemical and serologic al tests were positive in the multiplex PCR. EHEC O157:H7 was identifi ed from all of 70 experimentally inoculated ground beef and milk sampl es which had initial inocula of 4 to 9 CFU/g (ml) and were subjected t o a 6-h enrichment culturing. The multiplex PCR procedure could be ver y useful for routine examinations of food samples for the presence of EHEC O157.