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United States Department of Agriculture

Agricultural Research Service

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Location: Molecular Characterization of Foodborne Pathogens Research

2011 Annual Report

1a. Objectives (from AD-416)
The long-term objectives of this project are to develop, validate, and implement new technologies and systematic approaches for detection of microbial and chemical contamination of foods. Goals will be accomplished by utilizing multi-disciplinary research teams that involve food scientists, microbiologists, molecular scientists, and agricultural, biological, and electrical engineers.

1b. Approach (from AD-416)
The approach focuses on four main components including separation, detection, identification, and quantification of target microorganisms from food matrices.

3. Progress Report
Our approach involves the development of a bioseparation technology that can separate and concentrate pathogenic microorganisms from food matrices and systems, and the development effective methods for the detection and quantification foodborne pathogens. The bioseparation technology concentrates cells directly via a sequential filtration process, achieving a 1000-fold concentration of cells in less than 60 min. The filtration system was improved this year by optimizing filter materials, hollow fiber membrane designs, enzyme treatments before pre-filtration of food-derived samples, and cleaning and sterilization procedures. Results indicate the possible re-use of the membrane up to 20 times, thereby reducing both cost and manual operations associated with recovery of concentrated microbial samples. The filtration device was used successfully to separate and concentrate pathogens from complex foods (i.e., Salmonella from chicken). A wide variety of platforms appropriate for detection of the concentrated pathogens were also studied One detection method uses a microfluidic biochip that incorporates dielectrophoresis (DEP) to further concentrate bacteria, as well as for growth and lysis of the bacteria, and detection of pathogen-specific genes using the polymerase chain reaction (PCR). Investigations this year demonstrated the successful capture and detection of L. monocytogenes from a mixed microbial population on the biochip platform, and DEP enhanced L. monocytogenes capture. Label-free detection of PCR products was examined with electrically-based detection to aid in both detection and quantification. This methodology had an estimated detection limit of around 100 cells/mL. A second system called “BARDOT” (Bacterial Rapid Detection using Optical Scattering Technology) uses light scattering techniques to differentiate and classify bacterial colonies grown on Petri-dishes. This year, the database of scattering images from different pathogens was expanded, and BARDOT was able to differentiate and classify 26 different types of Salmonella and seven common types of Shiga-toxin producing E. coli. To reduce total detection time from food samples, an approach to detect micro-colonies was initiated, and BARDOT could differentiate micro-colonies of Salmonella, E. coli, and L. mono after only 8-12 h of growth. A third system was the design and synthesis of novel fluorescence-based DNA microarray probes for the PCR-based detection of three target pathogens; Salmonella enterica, Listeria monocytogenes, and E. coli O157:H7. A third generation prototype PCR chamber was constructed. Finally, methods employing infrared spectroscopy were improved this year by determining how spectroscopic differentiation relates to cell surface characteristics, how these structures vary with conditions and treatments (including differentiation of live and dead cells), how the techniques could be applied to detect pathogens in food systems, and for classification (i.e., typing) of strains of L. monocytogenes, E.coli O157:H7, and Salmonella.

4. Accomplishments

Last Modified: 2/23/2016
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