Food Safety Research Information Office
Title:Detection and Typing of Foodborne Pathogens
Objective:

Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens.

Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements.

Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification.

Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a naïve library of biorecognition elements for bacterial typing-An alternative to "molecular typing."

More Info:
Approach: 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.
Funding Source:United States Department of Agriculture (USDA), Agricultural Research Service (ARS)
Type:Appropriated
Start Date:2011
End Date:2016
Project Number:1935-42000-071-00
Accession Number:421013
Institutions:USDA/ARS - North Atlantic Area
Investigators:Bhaduri, Saumya
Brewster, Jeffrey
Gehring, Andrew
He, Yiping
Paoli, George
Project Reports:2013 Annual Report
2012 Annual Report
2011 Annual Report
Pending USDA ARS Publications (NP 108):
Published Journal
Articles USDA
ARS (NP 108):
Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples
Suo B, He Y, Irwin PL, Gehring AG .
Food Anal Methods. 2013 Oct;6(5):1477-84.
A high-throughput antibody-based microarray typing platform
Gehring A, Barnett C, Chu T, Debroy C, D'Souza D, Eaker S, Fratamico P, Gillespie B, Hegde N, Jones K, Lin J, Oliver S, Paoli G, Perera A, Uknalis J .
Sensors. 2013 May 3;13(5):5737-48.
Food Safety Categories:Government Policy and Regulations
Methodology
Farm-to-Table categories:Food processing
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