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United States Department of Agriculture

Agricultural Research Service

Title: Rapid Detection of Outbreak Escherichia Coli 0157 Adn Salmonella in Alfalfa Sprouts by Immunomagnetic Capture and Time-Resolved Fluorescence

Authors
item Tu, Shu-I
item Golden, Marsha
item Fett, William
item Gehring, Andrew
item Irwin, Peter

Submitted to: Journal of Food Safety
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 14, 2003
Publication Date: March 14, 2003
Citation: TU, S., GOLDEN, M., FETT, W.F., GEHRING, A.G., IRWIN, P.L. RAPID DETECTION OF OUTBREAK ESCHERICHIA COLI 0157 ADN SALMONELLA IN ALFALFA SPROUTS BY IMMUNOMAGNETIC CAPTURE AND TIME-RESOLVED FLUORESCENCE. JOURNAL OF FOOD SAFETY.

Interpretive Summary: Since 1995, raw alfalfa sprouts have emerged as a recognized source of foodborne illness in the United States by the Food and Drug Administration. Both Salmonella and E. coli O157 outbreaks associated with alfalfa sprouts have been reported. With annual sales of alfalfa sprouts in U.S. approaching $250 million dollars, the demand on the safety of this popular salad food is also increased. In this study, we have developed a new approach to detect E. coli O157:H7 and Salmonella in the sprouts germinated from seeds containing as low as 4 CFU of the pathogens per gram of the seeds. After an enrichment for 4 hours, cells of E. coli O157:H7 and Salmonella from spent irrigation water or sprouts were captured by specific antibodies coated on magnetic beads. Captured E. coli and Salmonella were further tagged by metal europium and samarium, respectively. From the unique fluorescence properties of the two metals, we were able to determine the presence of one or both bacteria. The information is useful for researcher and/or engineers to design an automated process for multiple pathogen detection in sprouts.

Technical Abstract: Commercially available alfalfa seeds were inoculated with low levels (~ 4 CFU/g) of pathogenic bacteria. The inoculated seeds were then allowed to sprout in sterile tap water at 22 0C. After 48 hours, the irrigation water and sprouts were separately transferred to bovine heart infusion (BHI) media. The microbes in the BHI samples were enriched for 4 hours at 37 oC and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E.coli O157 and/or Salmonella in the enriched media. Separation and concentration of captured pathogens were achieved using magnetic separators. IMB captured E. coli O157:H7 and Salmonella spp formed sandwiched complexes with europium (Eu) labeled anti-E. coli O157 antibodies and samarium (Sm) labeled anti-Salmonella antibodies, respectively. After washing, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent chelates. The specific time-resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and many Salmonella enterica from the seeds inoculated with ~ 4 CFU/g of the pathogens. Non-targeted bacteria, e.g., Aeromonas and Citrobacter exhibited essentially no cross reactivity. Since the pathogen detection from the sprouts was achieved within 6 hours, the developed methodology could be use as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella enterica in this popular salad food.

Last Modified: 11/27/2014
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