DNA AMPLIFICATION USING TABLETED PCR REAGENTS FOR IDENTIFICATION OF ESCHERICHIA COLI O157:H7 ISOLATED FROM FOODS1

Abstract

A DNA amplification method using tableted multiplex polymerase chain reaction (PCR) reagents was developed for identification of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food samples. A suspect colony from MacConkey sorbitol agar containing 5‐bromo‐4‐chloro‐3‐indoxyl‐6‐D‐glucuronide (MSA‐BCIG) was used to perform the 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. This method detected 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 950 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 PCR. After a 6‐h enrichment, EHEC O157:H7 was identified from all experimentally inoculated ground beef and milk samples which had initial inocula of 4 to 9 CFU/g (mL). This method offers significant advantages in terms of technical simplicity and reproducibility of test results over conventional PCR and other assays for detecting E. coli serotype O157 and should be suitable for use in routine examination of food and other samples for the presence of the pathogen.