DEVELOPMENT OF PRIMERS FOR DETECTION OF MULTIPLE CERVID SPECIES IN ANIMAL FEED

Abstract

ABSTRACT Polymerase chain reaction (PCR) detection of mammal or ruminant tissue is used for ensuring compliance with animal feed regulations for BSE protection. In the event of a positive result using the group‐specific primers for mammal or ruminant, it is necessary to identify likely species sources of the contamination. To investigate contaminated species tissue, species‐specific primer can be used such as primers for cattle, sheep or deer. Although primers for single cervid species were reported, there are risks such as false negative result or non‐availability for feed, because they are not designed for inspection of feed and for detection of multiple cervid species. In this study, we have developed primers to detect multiple cervid species in feed. The primers we developed were designed from the whole mtDNA sequences of eleven cervid species. This primer set created 133‐bp DNA fragment from various deer mtDNA, and no amplification were observed from nontarget species, several feed materials and formula feed at the same length of the target PCR product. The detection limit was 0.1 pg of the mtDNA. PRACTICAL APPLICATIONBecause existing PCR primers for cervid species are designed to detect specific cervid species, they may produce false negative results when they are used for detecting deer in general. Moreover, animal feed contains various kinds of materials and some of them may become a inhibitor of the primers. The PCR method in this study is available to be used for an investigation for the contaminated cervid species in feed or feed materials. To avoid the false negative result, a consensus sequence for designing group‐specific primers was created from 11 cervid mtDNA sequences, using a computer program. This primer set differentiates various cervid species from non‐cervid species in feed.