Development of genetic tests for the rapid identification of species in foods.

The aim of the project was the development of DNA based tests that could be used to identify and distinguish common commercial meat species. The emphasis of the work was on development of a method that would be successful for cooked and highly processed foods. The potential of the PCR (polymerase chain reaction) as the basis of such a test was examined. The DNA sequence chosen as the target for PCR amplification was mitochondrial DNA (mtDNA). DNA sequence from a region of the mtDNA called the control region was obtained for cattle, sheep, pig, goat and chicken by DNA sequencing and from the literature. Using this information, species-specific PCR primers pairs were developed that allowed all the above species to be identified and distinguished. Amplification products were specific, showing no cross-reaction with other species, and were of a unique size for each species. In addition, multiplex PCR, using mixtures of species-specific primer pairs, allowed identification of several species in one PCR reaction. Amplification was successfully achieved from DNA extracted from blood, raw meat and food samples including cooked and highly processed foods and products. To assess the consistency of the species-specific primers, amplification was carried out on numerous individuals and breeds of particular species; amplification was successful for all individuals tested. DNA sequencing of the control region of individual animals was also carried out to assess the level of polymorphism within domestic meat species. These experiments showed that DNA polymorphism was low in domestic species and suggested that a species-specific PCR test based on the mtDNA control region would be consistent. The complete sequence of sheep mtDNA control region was obtained. The sheep control region is characterised by the presence of an area of repeated DNA consisting of four seventy five base pair repeats. Alignment of the control region sequence with other domestic species revealed that the putative mitochondrial control element CSB 1 is well conserved in these species. However, the control elements CSB 2 and 3 are not well conserved even in the closely related species of sheep, goat and cattle.