My. Deng et Pm. Fratamico, A MULTIPLEX PCR FOR RAPID IDENTIFICATION OF SHIGA-LIKE TOXIN-PRODUCING ESCHERICHIA-COLI O157-H7 ISOLATED FROM FOODS, Journal of food protection, 59(6), 1996, pp. 570-576
For rapid and specific identification of enterohemorrhagic Escherichia
coli (EHEC) serotype O157:H7 isolated from food samples, experimental
conditions for a multiplex polymerase chain reaction (PCR) were optim
ized and a multiple digoxigenin (DIG)-labeled oligonucleotide probe hy
bridization (DLOPH) assay was developed. A suspect colony from MacConk
ey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-beta-D-glucuron
ide (MSA-BCIG) was used for the multiplex PCR. Three different DNA seq
uences off. coli O157:H7 were amplified simultaneously in the PCR: a s
pecific fragment of an attaching and effacing gene (eae gene), conserv
ed sequences of Shiga-like toxins (SLT) I and II, and a fragment of th
e 60-MDa plasmid. The identities of PCR products were confirmed by hyb
ridization using DIG-labeled internal oligonucleotide probes and color
imetric detection with anti-DIG Fab fragments conjugated to alkaline p
hosphatase. This method yielded positive results with all reference st
rains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, an
d O157:H-, and negative results were obtained with all strains of nont
oxigenic E. coli serogroup O157, other serotypes of E. coli, and other
bacterial species. The detection limit of the method was 65 colony-fo
rming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7
isolated from meat samples and identified by biochemical and serologic
al tests were positive in the multiplex PCR. EHEC O157:H7 was identifi
ed from all of 70 experimentally inoculated ground beef and milk sampl
es which had initial inocula of 4 to 9 CFU/g (ml) and were subjected t
o a 6-h enrichment culturing. The multiplex PCR procedure could be ver
y useful for routine examinations of food samples for the presence of
EHEC O157.