"Inadvertent contamination of foods with harmful microorganisms can result in multiple problems including loss in productivity, expenses related to healthcare, investigation, litigation, destruction of vast quantities of agricultural products, and loss of human life."

Multiplexed detection of pathogens using fluorescence resonance energy transfer in spatial detection format

Investigator: Bruce Applegate (Department of Food Science)

Project Report 2005 - 2006

» Download Project Report 2005 - 2006

Project Rationale

Inadvertent contamination of foods with harmful microorganisms can result in multiple problems including loss in productivity, expenses related to healthcare, investigation, litigation, destruction of vast quantities of agricultural products, and loss of human life. To streamline efforts in the circumvention of food poisoning-related incidents, FSIS requires a fully automated testing system for the rapid throughput analysis of foods for contaminant pathogens (E. coli O157:H7, Salmonella, Listeria spp., etc.). Ideally, the testing platform will be a technician-operated instrument that can simultaneously screen food samples for the presumptive presence of multiple bacteria and, if desired, confirm their presence and characterize the pathogens through identification of virulence-related or other genes. This approach would ultimately eliminate the need for the time-consuming conventional cell culture/isolation/confirmation procedures currently used. The specific objective of this project is the development of a nucleic acid microarray for the detection of multiple PCR products for the identification of foodborne pathogens E. coli O157:H7, Salmonella spp. and L. monocytogenes, based on molecular beacon technology. This project leverages capabilities in DNA microarray technology to develop a gene-specific assay that does not require costly labeling and purification methods to detect the presence of the target gene.

Project Objectives

  • Construct an initial prototype gene array consisting of four marker genes for E. coli O157:H7 utilizing molecular beacon probes immobilized on a glass slide. (2005)
  • Determine hybridization conditions and evaluate the prototype beacon array utilizing amplified target genes.(2005)
  • Contruct microarrays containing four targets from Salmonella spp. and L. monocytogenes from previously constructed target probe and amplicon sequences along with microarrays including all three sets of probes for the simultaneous detection of E. coli O157:H7, Salmonella spp. and L. monocytogenes. (2006)
  • Design and develop probe and amplicons for Campylobacter, Clostridium, and S. aureus. (2006)
  • Develop and integrate a hybridization and PCR control into the array to provide quality assurance. (2006)
  • Construct arrays for multiple organisms and evaluate for hybridization specificity. (2006)
  • Evaluate previously developed multiplex PCR reactions for simultaneous amplification of multiple pathogen targets utilizing the developed microarrays. (2006)

Project Highlights

This project focuses on the integration of the microarray work with the previously developed spatial array format. A DNA hybridization-based optical biosensor for the detection of foodborne pathogens was developed with virtually zero probability of a false negative signal. This portable, low-cost and real-time assaying biosensor utilizes the color-changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled biosensor exploits the target hybridization-induced change of fluorescence color due to the Förster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon. Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows one to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection that is characteristic of the fluorophore-quencher beacon for which bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. The two-color design allows us to quantify the concentration of the target DNA in a sample down to ≤0.5 ng/μl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.

Annual Report

Investigator