A Multiplex PCR for Rapid Identification of Shiga-Like Toxin-Producing Escherichia coli O157:H7 Isolated from Foods‡

Abstract

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.