微滴式数字聚合酶链式反应对香肠制品中鸡、猪、牛源性成分的定量分析

为实现肉制品中动物源性成分的定量检测，基于微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR），以鸡的转化生长因子β-3基因、猪的朊蛋白基因和牛的生长激素基因为靶基因，建立香肠制品中鸡、猪、牛源性成分的定量检测方法。结果表明：所建立ddPCR方法特异性强，能特异性检测相应的鸡、猪、牛源性成分；根据肉粉质量（mg）与DNA质量浓度（ng/μL）、DNA质量浓度（ng/μL）与基因拷贝数浓度（copies/μL）之间的线性关系，得到鸡肉粉、猪肉粉和牛肉粉质量（x1）与基因拷贝数浓度（ y 2） 之间的关系式为： 鸡：x1=（n*y2）/273.946 - n/886.557 + 0.216； 猪：x1=（n*y2）/64.950 + n/60.644 + 0.215； 牛：x1=（n*y2）/62.839 + n/12.646 + 1.218（n为稀释倍数）；基于建立的ddPCR方法对64 份市售不同种类香肠样品进行检测，在1 份原料标识仅为“猪肉”的香肠中检出鸡源性成分含量为18.68%。本研究建立的ddPCR方法能够实现香肠中鸡、猪、牛源性成分的准确定量检测，并可以此判断“蓄意掺假”或“无意带入”。

Quantitative Analysis of Chicken-, Porcine- and Bovine-Derived Ingredients in Sausage Products by Droplet Digital Polymerase Chain Reaction

A droplet digital polymerase chain reaction (ddPCR) assay based on the transforming growth factor beta 3 gene of chicken, the porcine prion protein gene and the bovine growth hormone gene as the target genes were developed for the quantitative detection of chicken-, porcine- and bovine-derived ingredients in sausage products. The results showed that the established assay was highly specific for the detection of chicken-, porcine- and bovine-derived ingredients, but not for other tested animal components. Based on the linear relationship of DNA concentration (ng/μL) with meat mass (mg) and the number of gene copies per unit volume (copies/μL), the linear equations between meat mass (x1) and the number of gene copies (y2) were established as follows: x1=（n*y2）/273.946 - n/886.557 + 0.216 for chicken；x1=（n*y2）/64.950 + n/60.644 + 0.215 for pork；x1=（n*y2）/62.839 + n/12.646 + 1.218 for beef (where n is dilution factor). The ddPCR assay was used for the quantitative detection of 64 samples of different kinds of comercially available sausages. It was found that one of these samples, labelled as containing only pork, contained 18.68% chicken-derived ingredients. The ddPCR assay could allow accurate and quantitative analysis of chicken-, porcine- and bovine-derived ingredients in sausage, and could be used to discriminate intentional and unintentional adulteration.