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United States Department of Agriculture

Agricultural Research Service

Title: Time-Resolved Fluorescence Detection of Shiga-Like Toxins Produced by Escherichia Coli 0157 and Non-0157 in Ground Beef

Authors
item Tu, Shu-I
item Golden, Marsha
item Paoli, George
item Gehring, Andrew
item Gore, Mitchell - POLYSCIENCES, INC.

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 7, 2004
Publication Date: September 22, 2004
Citation: Tu, S., Golden, M., Paoli, G., Gehring, A.G., Gore, M. 2004. Time-resolved fluorescence detection of shiga-like toxins produced by escherichia coli 0157 and non-0157 in ground beef. Meeting Abstract.

Technical Abstract: Foods contaminated with Shiga like toxin (SLT)-producing Escherichia coli (STEC) pose a significant threat to public health because ingestion of small numbers of viable organisms (<100 cells) may cause hemorrhagic colitis and may lead to life-threatening sequelae such as hemolytic-uremic syndrome or thrombotic thrombocytopenic purpura in susceptible individuals. Conventional methods for detecting SLTs in foods and fecal samples include a vero cell-based cytotoxicity assay, an antibody-based direct detection assay and an indirect enzyme-linked immunosorbent assay. However, the detection limit of these methods is greater than 1 ng/mL. An immunomagnetic bead time-resolved fluorescence assay (IMB-TRF) assay was developed for the detection of Shiga-like toxins I (SLT I and SLT II). The method could detect 5 pg/mL of SLT II and 50 pg/mL of SLT I. To detect SLTs from STECs, twenty five gram ground beef patties were inoculated with 1 CFU/g of O157 or non-O157 STEC, stored at 7C for 5 h and then incubated with 100 mL of EC broth at 37C and 140 rpm shaking for 20 h. Ofloxacin was added to the ground beef at a final concentration of 10 µg/mL after 20 h of incubation to enhance toxin production. After four more hours of incubation, samples were removed and centrifuged for 10 m at 5000 rpm. 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 that were then recombined with extra-cellular SLTs present in the supernatant. Immunomagnetic beads coated with monoclonal antibodies specific to SLT I and SLT II were applied to capture and concentrate the toxins and captured toxins were labeled with lanthanide-tagged anti-toxin antibodies to form sandwiched complexes prior to detection by a time-resolved fluorescence (TRF) reader. TRF pathogen detection levels, defined as signals greater than or equal to 2X un-inoculated samples, were achieved in all STEC inoculated samples and no cross-reactivity was observed with either nonpathogenic microflora present on the meat or non-toxin-producing Escherichia coli K12 inoculated samples.

Last Modified: 10/1/2014
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