一种基于双重ddPCR的肉制品中牛源性成分的定量分析方法

将低价肉类原料掺入高价肉制品是一种典型的肉类掺假方式，为准确鉴别牛肉及牛肉制品的真伪，建立一种基于双重微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）的牛源性成分定量分析方法。通过对14 种动物的Beta-actin单拷贝基因序列进行分析，设计牛源性特异性引物、探针与动物源性成分通用引物、探针并建立双重ddPCR体系，推导出牛源性成分质量与其特异性扩增拷贝数之间的计算公式，对牛源性成分进行定量检测该方法得到牛源性成分质量M牛（mg）与其特异性扩增拷贝数浓度C牛（copies/μL）之间的计算公式为M牛=0.033C牛＋2.37，对已知牛肉质量的混合肉样品与不同部位的牛肉样品进行检测，结果显示牛肉定量检测值与实际质量基本一致。同时拷贝数相对含量可辅助判断肉制品中是否存在非牛源性的其他动物源性成分掺假。对市售样品的检测发现，存在目标肉样含量不达标以及不同程度的动物源性成分等掺假现象，说明该检测方法可为牛肉制品掺假量化判定和混合源性产品分级鉴定提供技术支撑。

Quantification of Bovine-Derived Ingredients in Meat Products by Duplex Droplet Digital Polymerase Chain Reaction

One typical way of meat adulteration is by adding low-priced meat ingredients into high-priced meat products. In this study, a duplex droplet digital polymerase chain reaction (ddPCR) method was developed to accurately identify bovine-derived ingredients in meat products. By analyzing the single-copy β-actin gene sequences of 14 animal species, bovine-specific primers and probes as well as universal primers and probes for animal-derived ingredients were designed to develop a duplex ddPCR system. The formula to calculate the relationship between the mass of bovine-derived ingredients (M, mg) and the number of copies of specifically amplified genes (C, copies/μL) was established as follows: M = 0.033C + 2.37. The results of this method for mixed meat samples containing a known amount of beef and different beef cuts basically agreed with the actual values. Moreover, the relative copy number was helpful to judge whether beef products could be adulterated with non-bovine-derived ingredients. The developed method was successfully applied to detect the adulteration of commercial beef products. We believe that this method will provide technical support for the quantitative detection of adulterated beef products and grading and identification of meat mixtures from different animal specices.