SF6033 - Development of a Robust DNA Preparation Method to Enhance Simultaneous Detection of Multiple Pathogens in Foods by a Microarray-Based Assay

Researcher:

Dr. Shu Chen, Laboratory Services Division, University of Guelph

Objectives:

1. To integrate disparate techniques into a simple and unified DNA isolation method for handling multiple pathogens of various differences in various food matrices.
   

2. To develop an effective DNA enrichment method based on the new multiple displacement amplification (MDA) technology to make compensation for low concentrations of slow-growing pathogens or pathogens that can not be enriched in food samples.
   

3. To determine the feasibility of using the MDA technology directly with our existing microarray without PCR for food testing.

Expected Benefits:

• A robust DNA extraction and enrichment method for food samples, including poultry, red meat, milk and vegetables, which can be used as a front end for PCR/microarray-based assays.
  

Summary of Research Results:

The researchers investigated a new DNA preparation method based on the multiple displacement amplification (MDA) technology, and developed an effective sample preparation, "universal" DNA extraction and enrichment method for food and water testing. The method was successfully integrated into the microarray assay, and evaluated using artificially and naturally contaminated food and water samples. The results demonstrated that the integrated assay can be used for the simultaneous detection of Campylobacter, Salmonella, S. typhimurium DT104, Shiga toxin-producing E. coli, E. coli serotype O157:H7, Listeria monocytogenes, Giardia intestinalis, Cryptosporidium parvum and Cyclospora cayetanensis. The assay was able to detect as low as single bacterial cell or 4-40 parasite cells in one mL of pre-enriched food samples. As compared to the conventional cultural methods upon testing 162 samples, the percentages of culturally positive samples that were identified as positive by the new assay were 100% for Campylobacter, 100% for Salmonella, 94% for L. monocytogenes and 100% for Shiga toxin-producing E. coli; the percentages of culturally negative samples that were identified as negative by the new assay were 88% for Campylobacter, 96% for Salmonella, 85% for L. monocytogenes and 90% for Shiga toxin-producing E. coli.

The results suggest that the unified sample preparation, DNA isolation and MDA method developed in this project significantly enhanced the microarray-based detection, and the integrated assay was more sensitive than the culture methods. The assay can be used as a screening tool for the rapid, sensitive and cost-effective detection of the target pathogens in food and water samples upon further validation. The DNA preparation method can be extended as a front end to other nucleic acid-based detection assays for testing food and water samples.

Availability of such an assay will allow diagnostic labs to provide faster and more cost-effective results to food industries and government agencies. This will also allow scientists to conduct risk assessment studies in a systematic and cost-effective manner in support of monitoring and surveillance activities and enforcement of regulatory programs. This will help to ensure that the Ontario agri-food industries remain competitive in providing safe foods to today's food safety conscious consumers.