Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 10, 2003
Publication Date: November 9, 2003
Citation: YU, L.S., REED, S.A., TARKKINEN, P., TU, S. DETECTION OF ESCHERICHIA COLI 0157:H7 IN FOOD BY A MICROPLATE SANDWICH IMMUNOASSAY USING TIME-RESOLVED FLUOROMETRY. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. v. 11. p. 133-143. 2003. Interpretive Summary: Food industry and public health microbiologists need reliable and rapid methods to screen high-risk foods for E. coli O157:H7. We have developed a sensitive and rapid method for the detection of E. coli O157:H7, which combines microplate sandwich hybridization immunoassay using Europium chelate-labeled antibodies and time-resolved fluorescence detection. Using gthese methods as few as 10 cells spiked beef hamburger or apple cider samples could be detected after a six-hour enrichment. Food industries could benefit from the application of time-resolved fluorescence technology with high throughput, which reduces the time required to analyze food for pathogenic bacteria and, hence, enables processors to respond more rapidly to situations involving contaminated food products.
Technical Abstract: Escherichia coli O157:H7 has been identified as a causative agent in outbreaks of hemorrhagic colitis and hemolytic uremic syndrome involving ground beef and apple cider. A microplate sandwich immunoassay for the detection of E. coli O157:H7 from food was developed utilizing time- resolved fluorometry. Biotin-labeled, polyclonal anti- E. coli O157:H7 antibodies immobilized on streptavidin-coated microtiter plates were used to capture the bacteria. The bound antigen-antibody complex was then detected using the same antibodies labeled with europium chelates. The effects of capture antibody concentrations (2-5 ug/ml), immobilization procedures (25 degrees C, 1 h or 4 degrees C, overnight) and addition of 0.1% Tween 20 to assay buffer were studied during assay development. The detection threshold for the assay developed is between 1000 to 10000 CFU/ml. The optimized assay was further tested in ground beef and apple cider samples spiked with E. coli 0157:H7. The detection limit was less than 10 CFU/g of ground beef and about 10 CFU/ml of apple cider after a 6 h enrichment. The assay developed could constitute as an effective procedure for sensitive screening of E. coli O157:H7 in food with a total turn-around time of less than 8 h.