Use PCR to detect pathogens.

The diversity of foodborne pathogens continues to increase. This is partly due to environmental stress factors, including food preservation processes. These techniques may lead to changes in pathogen genotypes and the resistance or virulence of bacteria. This makes detection and identification of the pathogens a complex task that requires better approaches. For example, a U.S. survey has shown that only 18% of 76 million illnesses every year are caused by known pathogens.

As you may know, the polymerase chain resaction (PCR) is a sensitive molecular genetics technique in which the DNA of a single cell, treated with polymerase enzymes, is induced to replicate many times. This enables the DNA to be amplified in sufficient quantities to permit genetic analysis. PCR methods are extremely sensitive and specific to bacteria. They are based on the multiplication of the isolated pathogen DNA and the identification by electrophoresis of the DNA in the microbes. Even though PCR-based analytical kits are already on the market, standardization and validation of routine analysis are needed.

Scientists in Europe are attempting to validate a simple method for purifying DNA from bacterial cultures. They also want to establish a central collection of certified DNA sample materials and a databank containing key food-pathogen DNA sequences. The researchers would like to develop standardized reagents and will attempt to validate certain pre-PCR sample treatment methods.

This project involves a consortium of 35 institutes, companies and universities from across Europe. Researchers will devise standardized PCR-based detection methods for five major pathogens: S. enterica, thermophilic Campylobacter spp., enterohemorrhagic E. coli (EHEC), L. monocytogenes and Y. enterocolitica. The three-year project entails producing certified DNA material, preparing a thermocycler validation guideline and performing PCR trials. Another important focus will involve the development of automated detection methods.

The most important outcome so far has been the development of a guideline and a biochemical kit for validating different types of thermocyclers. Also, a simple method for purifying DNA from bacterial cultures has been developed. Reference DNA material has been produced that will be made available. An online PCR database is currently under construction. The databank will list strains for specificity testing.

Further information. Jeffrey Hoorfar, Danish Veterinary Institute, Bulowsvej 27, DK-1790 Copenhagen, Denmark; phone: [phone]; fax: [phone]; email: [e-mail].