DNA AMPLIFICATION USING TABLETED PCR REAGENTS FOR IDENTIFICATION OF ESCHERICHIA-COLI O157-H7 ISOLATED FROM FOODS

A DNA amplification method using tableted multiplex polymerase chain r eaction (PCR) reagents was developed for identification of enterohemor rhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food sam ples. A suspect colony from MacConkey sorbitol agar containing 5-bromo -4-chloro-3-indoxyl-beta-D-glucuronide (MSA-BCIG) was used to perform the PCR. Three different DNA sequences of E. coli O157:H7 were amplifi ed simultaneously in the PCR: a specific fragment of an attaching and effacing gene (eae gene), conserved sequences of Shiga-like toxins (SL T) I and II, and a fragment of the 60-MDa plasmid. This method detecte d all reference strains of EHEC serogroup O157, including serotypes O1 57:H7, O157:NM, and O157:H, and negative results were obtained with al l strains of nontoxigenic E. coli serogroup O157, other serotypes off. coli, and other bacterial species. The detection limit of the method was 950 colony forming units (CFU) off. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemic al and serological tests were positive in the PCR. After a 6-h enrichm ent, EHEC O157:H7 was identified from all experimentally inoculated gr ound beef and milk samples which had initial inocula of 4 to 9 CFU/g ( mL). This method offers significant advantages in terms of technical s implicity and reproducibility of test results over conventional PCR an d other assays for detecting E. coli serotype O157 and should be suita ble for use in routine examination of food and other samples for the p resence of the pathogen.