Detection of Eschericia coli O157:H7 by fluorescence polarization assay and polymerase chain reaction.
It is recognized that cattle and other domestic animals can be a reservoir of pathogenic Escherichia coli, including serotype O157:H7. To contain this potential health hazard, the first step is the identification of the carrier animals. For these purposes, a rapid serological screening test, a fluorescence polarization assay (FPA) was developed and results obtained from a randomly selected cattle population as well as cattle immunized with E. coli O157:H7 were compared to those obtained with an indirect enzyme immunoassay (IELISA). To identify pathogenic strains in carrier animals, polymerase chain reactions (PCR) for Shiga-like toxins I and II were implemented using agarose electrophoresis. The sensitivity of the fecal extracted E. coli for Shiga-like toxin I and II was approximately 200 CFU per reaction using multiplex hot-start nested PCR. The sensitivity of the fecal extracted E. coli varied from approximately 5x10(2) to 2.5x10(3) CFU per reaction depending on the commercial kits used. The combination of the serological screening FPA and hot-start nested PCR confirmatory assays provided rapid identification of the pathogen.
Nielsen
K
K
Ontario Laboratories (Fallowfield), Canadian Food Inspection Agency, Ottawa, Ontario, Canada. nielsenk@inspection.gc.ca
Smith
P
P
McRae
H
H
Yu
W
W
Widdison
J
J
D000818:Animals; D002417:Cattle; D004269:DNA, Bacterial / Q000097:blood* / Q000302:isolation & purification*; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005243:Feces / Q000737:chemistry / Q000382:microbiology*; D005454:Fluorescence Polarization / Q000379:methods; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012680:Sensitivity and Specificity; D012698:Serologic Tests; D013997:Time Factors
D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic