SF6014 - New Technologies for Improving Real-Time PCR Methods for Detection of Food-borne Pathogens

Researcher:

Dr. Mansel Griffiths, Dept. of Food Science, University of Guelph

Objectives:

• To develop real-time PCR systems that can detect, differentiate and, where appropriate, enumerate Listeria spp., Salmonella spp., Campylobacter jejuni, E. coli 0157:H7 and total E. coli in a variety of samples.

Expected Benefits:

• Development of methods for the detection and enumeration of bacterial pathogens and indicator organisms that have sufficient sensitivity, specificity and speed to meet the requirements of the food industry, and the expectations of regulatory agencies.

Summary of Research Results:

It has been estimated that there are over 300,000 cases of foodborne illness in Ontario each year resulting in at least 34 deaths and a cost to the provincial economy in excess of $630 million. Not only do foodborne diseases impact on human health, but also trading nations like Canada risk losing global markets for foodstuffs if we cannot demonstrate that our products are safe. Thus, there is a critical need for Ontario to continue to develop new, rapid and reliable methods for the detection of foodborne pathogens. The aim of this project is to develop methods for the detection and enumeration of bacterial and viral pathogens having sufficient sensitivity, specificity and speed to meet the requirements of the food industry and the expectations of regulatory agencies. Such systems are essential for evaluating the safety of a product, validating safe food production and processing practices, providing information to assess the risk of illness arising from the consumption of a particular food, and even predicting shelf life.

The choice of testing method is determined mainly by regulatory standards and by cost-benefit analysis. Methods based on the unique genetic makeup of microbes allow economical and reliable detection of the pathogens that may be present in foods. One such method, called real-time PCR, offers the most timely, sensitive, and practical way of meeting new detection standards in short time frames. Although real-time PCR methods have been applied to foods, they have suffered due to inhibition of the assay by inhibitors present in the samples.

Thanks to funding by the Ontario Ministry of Agriculture and Food, we have developed real-time PCR methods for the detection of 4 common bacterial pathogens (E. coli O157:H7 (the Walkerton bug); Salmonella; Campylobacter; and Listeria monocytogenes) and 1 virus (Hepatitis A) that provide results within 24 h. The methods have been tested using a variety of food products relevant for the pathogen of concern such as red meat, poultry, fresh produce and sprouts. In addition, methods to improve extraction of DNA for use in real-time PCR assays have been investigated and an automated DNA purification system has been found to give good results. A simple concentration step involving filtration has also been developed for use in conjunction with real-time PCR assays and the combined method is capable of detecting 1 bacterial cell or 1 virus particle in the original food in less than a day. The introduction of these tests into the food industry will help insure that Ontarians continue to enjoy one of the safest food supplies in the world.