Time-resolved Fluorescence Detection of Shiga-like Toxins Produced by Escherichia Coli O157 and Non-o157 in Ground Beef1

An immunomagnetic bead time-resolved fluorescence assay (IMB-TRF) was developed for the detection of Shiga-like toxins I and II (SLT I and II) from Shiga-like toxin-producing Escherichia coli (STEC). Twenty-five gram of ground beef patties were inoculated with 1 CFU/g of O157 or non-O157 (STEC) stored at 7C for 5 h and incubated with 100 mL added EC broth at 37C and 140 r.p.m. shaking for 20 h. Ten mg/mL of ofloxacin was added to the EC broth after 20 h of incubation to enhance toxin production. After 4 more hours of incubation, samples were removed and centrifuged for 10 min at 5000 r.p.m. The supernatant was removed and saved and a bacterial protein extraction reagent was used to lyse the cells in the pellet and release intracellular SLTs (Fraction I) which were then recombined with extracellular SLTs (Fraction II) present in the supernatant. Immunomagnetic beads coated with monoclonal antibodies specific to Shiga-like toxins I and II were applied to capture and concentrate the toxins and captured toxins were labeled with lanthanide-tagged antitoxin antibodies to from sandwiched complexes prior to detection by a TRF reader. TRF pathogen detection levels, defined as signals ≥2¥ uninoculated samples, were achieved in all STEC inoculated samples and no cross-reactivity was observed with either nonpathogenic microflora present on the meat or nontoxin-producing E. coli K12 inoculated samples. Blackwell Science, LtdOxford, UKJRMJournal of Rapid Methods and Automation in Microbiology1060-3999Copyright 2004 by Food & Nutrition Press, Inc., Trumbull, Connecticut.December 2004124247258Original Article RAPID DETECTION OF SHIGA-LIKE TOXINS BY TRF S.-I. TU