Project Number: 8072-42000-071-00
Start Date: Apr 01, 2011
End Date: Mar 31, 2016
This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations.