Applications of time-resolved fluoroimmunoassay to detect magnetic bead captured Escherichia coli O157 : H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, strept avidin coated magnetic beads conjugated with biotin-labeled anti E. coli O1 57:H7 were used to capture the bacteria. ne bacteria were, at the same time , also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157: H7 antibody. The sandwiched bacterial complexes were then concentrated usin g a magnetic particle concentrator and washed to remove other solution comp onents. Upon addition of an enhancement buffer, the Eu-labels were then rel eased from the antibodies and chelated to nitrilo-triacetic acid (ATA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-ATA)(3)(TOP O)(2-3) micellar complexes. Delayed fluorescence associated with these comp lexes was measured and its intensity was used to estimate the original bact erial concentration spiked in hamburger. This approach was applied to detec t E. coli O157:H7 spiked in hamburgers. The results indicated this method i s able to detect similar to1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the app roach exhibited no or limited cross reactivity to Salmonella typhimurium, E . coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the develop ed approach may be used as a rapid screening procedure for E. coli O157 bac teria in foods.