2013 Annual Report
1a.Objectives (from AD-416):
The objective of this cooperative research project is to develop a multiplex superparamagnetic fluorescent microbead immunochemical assay for foodborne pathogens in food. Specifically, the scope of this project includes simultaneous detection and identification of the seven most common strains of Shiga toxin-producing Escherichia coli (STEC) based on O antigen: O157, O26, O111, O103, O121, O45, and O145. It also includes Listeria monocytogenes serotypes 1/2a, 1/2b, and 4b; and Salmonella enterica serovar Typhimurium. Foods of interest include ground beef, liquid egg, milk, and leafy greens. Performance characteristics of the product require sensitivity of at least one infectious dose of live bacteria per serving of food.
1b.Approach (from AD-416):
The overall design includes three components: (1) cultural enrichment, (2) sandwich ELISA format assay using monoclonal antibodies, and (3) a microbead array platform. The improvements to the cultural enrichment step will be accomplished using incremental adjustments of existing standard microbiological media and do not concern this Agreement. An enrichment step is necessary to provide sufficient sensitivity for an assay that must detect the presence of only a few organisms in a sample of several hundred grams of food. The sandwich ELISA format we will employ is commonly performed in microplate format, and is typical for microbead immunochemical assays developed for the Luminex platform. We will use monoclonal and polyclonal antibodies provided by SDIX. Each antigen assay will be deployed on a unique fluorescent labeled microbead set. Specificity and sensitivity will be confirmed using bacteria grown in culture broth. Validation will be performed by University of South Florida (USF) and FDA. For validation experiments pathogens will be spiked into foods, and the Lower Limit Of Detection will be determined for each organism, in the presence of other pathogens and typical background flora.
The research involves collaboration with Romer Technologies (who acquired the food safety division of SDIX), the University of South Florida, and the Food and Drug Administration. An antibody-based test was developed that determines which of the 7 most common types of Shiga toxin-producing E. coli (STEC) are present in a sample. This test gave the correct result on 78 of 79 strains of STEC strains that we tested. The antibody-based test and a gene-based test (developed by cooperators at FDA) were expanded to produce complementary 10-plex systems, each capable of detecting the 10 most common STEC. Validation of the assay for bacteria in culture is in progress, and validation experiments using spiked food samples will follow. Research progress reported addresses objective 1: "Develop new assays for bacterial toxins and their variants, using immunological and other methods, with emphasis on applicability to practical problems facing the food industry and regulatory agencies" of the parent project.