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United States Department of Agriculture

Agricultural Research Service

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Location: Molecular Characterization of Foodborne Pathogens

2012 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:

Progress was made on both objectives 1 and 2 and several associates subobjective, all of which fall under National Program 108, Food Safety, specifically contributing to Component 1: Food Contaminants and Problem Statement 1.C: Technologies for the Detection and Characterization of Microbial Contaminants of the 2011-2015 Strategic Action Plan. Our approach involves development of a bioseparation technology to separate and concentrate pathogenic microorganisms from food matrixes, as well as effective methods for the detection and quantification of foodborne pathogens. The bioseparation technology concentrates cells directly via a sequential filtration process, achieving a 1000-fold concentration of cells in less than 30 minutes. The filtration system was improved this year by optimizing sanitation procedures and software, such that a ‘hands-off’ operation cycle from cell concentration and recovery to cleaning steps can be completed within 2 hours. The filtration device was used successfully to separate and concentrate pathogens from complex foods and coupled to quantitative polymerase chain reaction (qPCR) analysis for detection of very low levels of Salmonella in chicken rinse water within 7 hours. A wide variety of platforms appropriate for detection of the concentrated pathogens were also studied. One detection method uses a microfluidic biochip to further concentrate bacteria, grow and break-open the bacteria, and detect specific pathogens using PCR. This year developments in field effect transistors (FETs) for single cell lysis and localized heating on the biochip were important steps to release and denature DNA from the bacteria cells as a precursor to PCR. Electrical detection of PCR products as an essential part of monitoring the reaction’s progress was also accomplished. 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. The database of scattering images from different pathogens continued to be expanded to include a wider variety of microorganisms, including molds, yeasts, and spoilage bacteria in addition to pathogenic bacteria, as well as more types of growth media. BARDOT was able to differentiate and classify 32 different types of Salmonella and seven common types of pathogenic Shiga-toxin producing E. coli. To reduce total detection time from food samples, detection of micro-colonies was optimized. BARDOT could differentiate micro-colonies of Salmonella, E. coli, and Listeria after only 8-12 h of growth on agar plates. Furthermore, BARDOT was equipped with multiple wavelength lasers to improve discriminatory power of scatter signatures and to detect microorganisms that are difficult to detect. A third system is a phage-based detection of E. coli O157:H7. Phage are bacterial viruses that infect specific bacteria. Genetic engineering tools were developed to manipulate the bacterial E. coli O157:H7 phage to produce a phage which causes the target bacteria to glow (luminesce) if infected. Initial experiments detected 1-5 cells in less than 12 hours using observable luminescence. Finally, methods employing Raman (infrared light) spectroscopy were improved this year by developing Raman probes and magnetic nanoparticles that are able to target pathogens and enhance Raman signals such that key pathogens (E. coli O157:H7, Salmonella and L. monocytogenes) could be detected at very low concentration.

Last Modified: 4/18/2014
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