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United States Department of Agriculture

Agricultural Research Service

Title: Applications of Time-Resolved Fluoroimmunoassay to Detect Magnetic Bead Captured Escherichia Coli O157:h7

Authors
item Tu, Shu-I
item Golden, Marsha
item Andreotti, Peter - PERKIN ELMER LIFE SCIENCE
item Yu, Linda
item Irwin, Peter

Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 1, 2001
Publication Date: N/A

Interpretive Summary: Contamination of pathogenic bacteria, e.g., E coli O157:H7 in foods may lead to serious public health concerns. To minimize possible outbreak of poisoning by this bacteria, sensitive and rapid detection techniques are needed to call for proper treatments to intervene further distribution of contaminated foods. Current available methods for detecting pathogenic bacteria are time consuming or of low specificity. In an attempt to develop specific and sensitive methods, we designed a new process in which cells of E. coli O157:H7 were captured by forming sandwiched complexes with two specific antibodies coated on magnetic beads and labeled with europium metal, respectively. The sandwiched complexes were concentrated and separated from other components with a magnet. The metal was then released to form highly fluorescent products that could be quantified. In application, hamburger spiked with about 1 cell of E. coli O157:H7 per gram mof meat, showed positive response after four and half hours of enrichment. With this method, the presence of low levels of E. coli O157:H7 may be determined within 8 hours. Furthermore, the approach can meet the high throughput requirement by the use of 96-well micro-plate format. The information is useful for researcher and/or engineers to design an automated process for detecting specific pathogens in foods.

Technical Abstract: A time-resolved fluorescence technique was developed to detect Escherichia coli in ground beefburger. Streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a non- fluorescent, europium (Eu)-tagged anti E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2- NTA)3(TOPO)2-3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to correlate with the original bacterial concentration. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method i able to detect ~ 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37 oC. Specificity studies indicated that the approach exhibited no cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.

Last Modified: 11/24/2014
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