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 enterotoxigenic 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-labile enterotoxin (LT) gene. The identity of the amplified products was confirmed by liquid hybridization using a horseradish peroxidase-linked internal oligonucleotide probe in a 96-well microplate coated with streptavidin. The final quantitation of the PCR products was performed by a colorimetric reaction. Under established conditions (including 1 min at 60 degrees C for primer annealing and extension in PCR cycles), this method detected 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 that 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. coli of porcine origin were detected while the results of other bacterial strains remained unchanged. In pure cultures, the detection 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 microliters was heated at 95 degrees 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.
Deng
M Y
MY
Eastern Regional Research Center, USDA, Agricultural Research Service, Wyndmoor, PA 19038, USA.
Cliver
D O
DO
Day
S P
SP
Fratamico
P M
PM
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000302:isolation & purification* / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D005516:Food Microbiology; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; C027538:heat-labile enterotoxin, E coli