My. Deng et al., ENTEROTOXIGENIC ESCHERICHIA-COLI DETECTED IN FOODS BY PCR AND AN ENZYME-LINKED OLIGONUCLEOTIDE PROBE, International journal of food microbiology, 30(3), 1996, pp. 217-229
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.