肉制品中牛源性成分Taqman荧光定量PCR检测法的建立

Establishment of Taqman fluorescence quantitative PCR assay for bovine derived ingredients in meat products

本实验根据牛线粒体差异序列设计特异性PCR引物和Taqman探针,并基于16S r DNA内参基因设计通用引物及Taqman探针,绘制并通过标准曲线确定样品中总DNA浓度以及牛源性成分DNA浓度,采用相对定量法测定肉制品中牛源性成分的质量分数。并通过特异性、灵敏度实验,及混合肉样回收率及市售肉制品检测,对本方法进行验证。结果表明,方法特异性强,可对牛肉DNA进行特异性扩增。最低检出限为10 pg/μL,具有较高的灵敏度。并根据回收实验可知,平均回收率为98.62%。可以为肉制品中牛肉含量的测定提供技术参考。

In this experiment,specific PCR primers and Taqman probes were designed based on the differential mitochondrial DNA sequences of bovine mitochondria,and based on the 16 S r DNA internal reference gene,universal primers and Taqman probes were designed. The total DNA concentration and DNA concentration of bovine source samples were determined by standard curve.Relative quantification method was used to determine the quality fraction of bovine source in meat products.The method was validated by specificity,sensitivity experiments,mixed meat recovery,and commercial meat testing.The results show that that method is specific and can amplify specific DNA of beef. The minimum observable limit with high sensitivity is10 pg/μL. According to the recovery test,the average recovery was 98.62%,which can provide technical reference for the determination of beef content in meat products.