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 optimized and a multiple digoxigenin (DIG)-labeled oligonucleotide probe hybridization (DLOPH) assay was developed. A suspect colony from MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-β-d-glucuronide (MSA-BCIG) was used for the multiplex PCR. Three different DNA sequences of E. coli O157:H7 were amplified simultaneously in the PCR: a specific fragment of an attaching and effacing gene ( eae gene), conserved sequences of Shiga-like toxins (SLT) I and II, and a fragment of the 60-MDa plasmid. The identities of PCR products were confirmed by hybridization using DIG-labeled internal oligonucleotide probes and colorimetric detection with anti-DIG Fab fragments conjugated to alkaline phosphatase. This method yielded positive results with all reference strains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, and O157:H ^- , and negative results were obtained with all strains of nontoxigenic E. coli serogroup O157, other serotypes of E. coli , and other bacterial species. The detection limit of the method was 65 colony-forming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemical and serological tests were positive in the multiplex PCR. EHEC O157:H7 was identified from all of 70 experimentally inoculated ground beef and milk samples which had initial inocula of 4 to 9 CFU/g (ml) and were subjected to a 6-h enrichment culturing. The multiplex PCR procedure could be very useful for routine examinations of food samples for the presence of EHEC O157.