应用微滴式数字聚合酶链式反应定量检测牛肉制品中的猪源性成分

参照GB/T 25165—2010《明胶中牛、羊、猪源性成分的定性检测方法 实时荧光聚合酶链式反应法》，以牛的生长激素（growth hormone，GH）基因和猪的朊蛋白（prion protein，PRNP）基因2 种核基因为靶基因合成特异性引物和探针。理论推导单位质量牛肉的GH基因与猪肉PRNP基因拷贝数之比为一固定值，通过微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）方法对该固定值进行检测和验证，并以该固定值对牛肉/猪肉人工混合样本和市售牛肉制品中的猪肉成分进行定量检测。结果表明：该方法具有良好的准确性及可重复性；牛肉中掺杂猪肉质量分数在5%～99%范围内时，检测结果绝对误差小于1.28%，变异系数小于6.5%，猪肉成分的回收率为99.09%～102.80%；20 份牛肉制品中，4 份检出猪肉成分，其中3 份的相对含量为29.19%～98.15%，1 份为0.12%（低于本方法检出限5%）。因此，ddPCR方法可以较好地应用于牛肉制品中掺杂猪肉成分的定量检测。

Quantitative Analysis of Pork in Adulterated Beef Products by Droplet Digital Polymerase Chain Reaction

According to the national standard GB/T 25165?2010, specific primers and probes targeting bovine growth hormone gene (GH) and porcine prion protein gene (PRNP), respectively were synthesized. The ratio of bovine GH gene copy number per unit mass of beef to porcine PRNP gene per unit mass of pork was derived to be a constant value theoretically, which was analyzed and verified by droplet digital polymerase chain reaction (ddPCR). This value was used to quantitatively detect added pork components in beef samples and pork components in adulterated commercial beef products. The results showed that ddPCR was highly accurate and repeatable. When beef added with 5%–99% was detected by ddPCR, the absolute error was less than 1.28%, the coefficient of variation less than 6.5%, and the recovery of pork 99.09%–102.80%. Pork components were detected in 4 out of 20 beef products; the content of pork was 29.19%–98.15% in three of the four and 0.12% (lower than the limit of detection of ddPCR, 5%) in the remaining one. The above results demonstrated that the developed ddPCR method is highly applicable for the quantitative determination of pork components in adulterated commercial beef products.