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Rapid Ultra-Concentration Technologies for Isolation of Pathogen DNA and RNA Markers from Foodstuffs for On-line Detection and Screening


Two key challenges that must be overcome prior to the implementation of practical biosensor and biochip technologies for testing of food and water samples for nucleic acids indicative of pathogenic contaminants are: (1) the provision of stable and reproducible sensing chemistries to achieve the desired device performance and allow manufacture, and, (2) to rapidly process on-line statistically representative samples (e.g. litres of fluid or grams of tissue) and deliver isolated target molecules to the sensing device in a small volume aliquot. We have developed a robust and reliable optical sensor technology for nucleic acid analysis. In a project funded by Genome Canada, we are currently developing a high sensitivity detection system prototype that will be capable of measuring nucleic acid targets directly from samples (i.e. without the need for enrichment of the nucleic acid concentration by amplification methods such as, for example, PCR). We are therefore well poised to address the second key challenge of rapid sample preparation that will permit near real-time automated analysis of pathogens in foodstuffs. On-line cell capture, ultrasonic disruption and microfluidic methods will be developed for rapid isolation and ultra-concentration of marker nucleic acids from food washes and surface wipes, and water, which will permit automated analysis for critical pathogens using our new nucleic acid biosensor technologies. By developing this missing link in the overall diagnostic method, rapid quantitative testing for multiple targets including bacteria, viruses and parasites at each link in the food supply chain may be realised. The technology will provide for inexpensive (per test) reliable screening that can be used in abattoirs, food processing plants and in-field, as identified as goals of the HACCP program.

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Expected Impact of Project Outcomes on Food Safety in Ontario:

It is estimated that over 80 million food-borne illnesses occur in North America each year, and that these can largely be attributed to E. coli, Salmonella, Campylobacter and Listeria monocytogenes (P.S. Mead et al., Emerg. Infect. Dis., 5: 607, 1999). Since Walkerton there have been a number of E. coli outbreaks attributed to the food supply. Public health officials have been anxious for a rapid monitoring system to assess food and water supply systems for microbial contamination. We have developed optical sensors that could rapidly detect interfacial nucleic acid hybridization with high selectivity. Results showed that genotyping hybridization assays could be done in minutes and that selective binding was observed with discrimination of single-nucleotide polymorphisms. Measurements were complete in 1-2 minutes, and the sensors have been demonstrated to be sufficiently robust that they may be reused for over 500 cycles of application (months of use) with good reproducibility (C.V. < 15 %) and no indication of degradation. The ultimate goal is approximately 1000 determinations per hour, at detection levels of 1000 molecules of nucleic acid target, i.e. single cells or a few cells when screening RNA. Our advances in detection technology have made it even more important to shift focus towards creation of methods to decrease the amount of time and effort necessary for sample collection and preparation. The problem is to take a sample of some liters in volume or grams in tissue mass, selectively collect organisms of interest while eliminating most of the sample volume, and then to produce a measurable target and bring this target in a concentrated small volume to a detector array. In partnership with Genome Canada, and an industrial partner, SafeGuard Biosystems, we intend to develop biosensor technologies that have the potential to provide the speed, selectivity and sensitivity required for effective analysis of food and water. <P> For more information, please visit the <a href="; target="_blank">Ontario Ministry of Agriculture, Food & Rural Affairs (OMAFRA) Food Safety Research Program</a>.

Krull, Ulrich
University of Toronto
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