应用通用引物量化检测鲜肉及其制品中羊源性成分

Quantitative Detection Method for Ovine-derived Materials in Fresh Meat and Meat Products Using Universal Primer

分别以牛、羊、猪、鸡、鸭、马线粒体细胞色素b基因为研究对象,设计羊源性成分特异性引物和探针,同时结合实时荧光定量PCR技术,引入16SrDNA内参基因校正羊种属特异性基因测定方法,建立快速、高通量的鲜肉及鲜肉制品中羊源性成分确证方法,实现科学准确的食品掺假量化判定技术体系。通过特异性、通用性及模拟混合样品的检测,对所建立方法进行验证。结果表明:建立的羊源性成分含量测定方法具有良好的特异性和通用性,且通过标准曲线的构建,呈现良好的线性关系均达0.996以上;通过羊种属特异性基因与16SrDNA内参基因Quantity值及校正系数,可以计算出样品中所含羊源性成份的质量百分比含量,经模拟混合样品的检测,回收率平均值到达96.25%,说明量化研究结果具有较高的准确性。

Targeting cytochrome b gene of mitochondrial DNA from beef,sheep,pork,chicken,duck,horse,the specific primers and TaqMan probes of sheep were designed. Combined with real-time PCR technology,the 16SrDNA reference gene was introduced,and the quantitative detection method of sheep in Fresh meat and meat products was established using reference gene correction by exogenous species-specific gene.The established method was validated through specific,universal detection,and analog mixed samples. The results showed a good specificity and a good universality.Through the construction of standard curve,both sheep-specific and u-niversal systems showed a good linear relationship（R2〉0.996）. By Quantity value and the correction coefficient of sheep species-specific gene and 16SrDNA reference gene,the mass percentage content of the sample con-tained in sheep-derived ingredients could be calculated. The PCR method showed a high accuracy and the aver-age recovery of sheep-derived ingredients in meat mixtures of simulation samples was 96.25%.