Improved Detection Techniques for Foodborne Pathogens: Multi-Plex Detection Platforms

Investigator: Bruce Applegate (Department of Food Science)

Project Report 2010 - 2011

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Project Rationale

Microbial contamination of food has become a mounting concern during the last decade, and rapid detection platforms are needed. Polymerase chain reaction (PCR) assays for bacterial detection have greatly reduced the time needed for detection compared to conventional microbiological and biochemical assays. For many years the successful PCR amplification of a target gene sequence was confirmed by the slow and tedious process of post-PCR gel electrophoresis. More modern PCR machines utilize fluorescently labeled DNA and optical sensors that allow the detection of PCR products in real-time. Multiplexed PCR relies on the amplification of multiple target sequences that are pathogen specific. The use of multiplexed PCR allows the detection of a suite of genes present in a pathogen, providing confidence in the identification and redundancy to help eliminate both false-negative and false-positive results.

The technology being developed by Center faculty is a fluorescence-based wavelength shift assay utilizing FRET (fluorescence resonance energy transfer) to detect hybridization of a set of target DNA sequences associated with target pathogens. The use of this spatial detection format (arrayed probes) will allow for the simultaneous detection of products from multiplexed PCR reactions more rapidly than could be achieved with electrophoresis. The spatial isolation of the probes allows for the simultaneous detection of a larger number of targets than would be feasible with current real-time PCR assays. Real-time multiplexed PCR relies on the use of a different pair of fluorophores for each molecular beacon due to the detection format. Currently there are few fluorophore pairs with distinct emission wavelengths to facilitate detection of large numbers of target DNA molecules. The technology we are developing will enable the construction of all of the beacons with a single fluorophore pair. The use of the same fluorophore pair for each beacon simplifies the design of the probes. This technology will be further expanded to include the use of a peltier chamber that can be cycled, creating an inexpensive multiplex PCR detection platform.

Project Objectives

  • Design and synthesis of FRET-based DNA microarray probes for the detection of target pathogens and to act as controls for PCR amplification and array hybridization.
  • Complete and validate a FRET-based DNA microarray for the detection of four target pathogens.
  • Complete and validate a prototype instrument that integrates peltier-based temperature controls with FRET-based microarray detection (excitation and emission).
  • Develop a prototype software control system.

Project Highlights

In prior years, we designed prototype reaction chambers to detect four specific genes associated with L. monocytogenes, Salmonella spp., and E. coli O157:H7. This year, a third generation PCR reaction chamber was constructed for detecting these foodborne pathogens and was modified to allow modular expansion for potential multifaceted applications. These applications include online monitoring of coliforms in potable water and swimming pools, and use in the emerging approach of personalized onsite medicine. This reconfigured system could also be used to control standard parameters associated with online monitoring of water samples (e.g., temperature, pH, free chlorine, redox potential, total dissolved solids, and turbidity) and multiplexed detection of microorganisms. Previously developed multiplex PCR detection assays were used to screen our current library of E. coli O157:H7 and Salmonella spp. isolates. All strains gave positive results for the four probes associated with each detection assay. These results continue to validate the robustness of the approach. The small footprint and inexpensive cost of the unit configured for food safety applications could greatly impact food safety by expanding and increasing foodborne pathogen testing.

""The small footprint and inexpensive cost of the PCR reaction chamber configured for food safety applications will greatly impact food safety by expanding foodborne pathogen testing.""

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