PCR-RFLP Identification of Prohibited Animal Derived DNA in Animal Feed

摘要

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.%わが国では，BSEの拡散防止のために，動物由来タン パク質の飼料への使用を規制している．この規制の実効性 を確保するために，飼料関係業者への立入検査が行われ， それに伴って収去された試料が分析されている．分析法の 1つとしてPCR法が用いられており，種特異的プライ マーによって規制動物種由来原料の有無を検出している． しかし，このPCR法は増幅断片の長さのみから検出の有 無を判断するため，陽性となった場合は配列確認などによ り非特異反応でないことの確認が望まれる．配列確認には シ￣クエンシングが最も確実な方法であるが，時間や費用 の点からルーチン検査に適していない．そこで本研究で は，短時間で簡便に同定確認を行うために，PCR＿RFLP による確認法を開発した．