ENTEROTOXIGENIC ESCHERICHIA-COLI DETECTED IN FOODS BY PCR AND AN ENZYME-LINKED OLIGONUCLEOTIDE PROBE

A polymerase chain reaction (PCR) and an enzyme-linked oligonucleotide probe hybridization assay were developed for the detection of enterot oxigenic Escherichia coli (ETEC) in ground beef, chicken, pork and raw milk. Two synthetic primers, one of which was biotinylated, were used in the PCR to amplify a fragment of the E. coli heat-table enterotoxi n (LT) gene. The identity of the amplified products was confirmed by l iquid hybridization using a horseradish peroxidase-linked internal oli gonucleotide probe in a 96-well microplate coated with streptavidin. T he final quantitation of the PCR products was performed by a colorimet ric reaction. Under established conditions (including 1 min at 60 degr ees C for primer annealing and extension in PCR cycles), this method d etected all 7 LT-producing E. coli pathogenic for humans, but did not detect all 7 LT-positive E. coli of animal origin, 3 E. coli strains t hat do not produce LT, and 9 other bacteria. Under less stringent PCR conditions (55 degrees C for annealing and extension), 2 strains of LT -producing E. coil of porcine origin were detected while the results o f other bacterial strains remained unchanged. In pure cultures, the de tection limit of the method was 1.4 colony forming units (CFU). Prior to PCR amplification, all food samples inoculated with an LT-producing ETEC, were subjected to enrichment in brain heart infusion broth for 8 h at 37 degrees C. From these cultures, 10 mu l was heated at 95 deg rees C for 10 min and directly used in the PCR. An initial inoculum of as few as 1.2 to 12 CFU of the LT-producing ETEC per 25 g (or ml) of food sample gave a positive reaction.