饲料及动物性食品中山羊源性和绵羊源性成分双重荧光PCR检测方法的建立

Development of A Dual Fluorescent Real-Time PCR for the Detection of Mitochondrial DNA of Goat and Sheep Mixed in Feed and Food Products

目的建立一种能同时检测饲料及动物性食品中的山羊和绵羊源性成分的双重荧光PCR方法,应用于动物源性饲料及动物性食品的快速检验。方法分别根据山羊、绵羊线粒体种间保守序列,设计合成针对山羊和绵羊的特异性引物及TaqMan探针,通过荧光PCR反应体系和反应条件的优化筛选,建立可同时检测饲料及动物性食品中的山羊和绵羊源性成分的双重荧光PCR方法。结果本研究所建立的双重荧光PCR检测方法可同时快速检测饲料及动物性食品中的山羊和绵羊源性成分,应用本方法检测山羊肉和绵羊肉模板的检出限均为10-5,与相应的单重荧光PCR方法的检出限一致,比国标法(GB/T20190-2006)的灵敏性高100倍。结论本研究建立的双重荧光PCR检测方法特异性好、敏感性强,适用于饲料、肉制品、乳制品等动物源性食品的快速检测。

Objective To develop a dual fluorescent real-time PCR assay for the simultaneous detection of goats and sheep-origined feed and goats and sheep-origined food products. Methods A dual fluorescent real-time PCR was developed using specific primers and TaqMan probes designed according to mitoehondrial DNA gene sequences, and screening by fluorescence PCR reaction system and the optimization of reaction conditions. Results The dual real-time PCR developed can detect less than 10-5 of goats and sheep-origined products, with the same as corresponding singlet fluorescence PCR method detection limit, and the 100 times higher than the sensitivity of the GB （GB/T20190-2006）.Conclusions The dual fluorescent real-time PCR was a specific and sensitive method for goat and sheep-origined materials in feed, and the animal-origined food products.