APPLICATIONS OF TIME‐RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

Abstract

A time‐resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, 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 nonfluorescent, 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 estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited 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.