基于实时荧光定量PCR和数字PCR的肉制品中牛源性成分检测

为了能够快速有效的检测出肉制品中牛源性成分,采用实时荧光定量PCR和数字PCR检测方法,检测肉制品中牛源成分。先依据牛肉单复制基因组特异区域,通过多序列比对设计特异性引物与探针,再利用苯酚-氯仿法提取标准品基因组DNA,确立实时荧光定量PCR和数字PCR两种方法的反应条件,测定DNA模版纯度及浓度可得实验提取的DNA溶液不含杂质,在此基础上构建牛源性成分荧光定量PCR标准曲线,检测两种方法的特异性、抗干扰性与肉制品中牛源性成分。分析实验结果可知,引物与探针在两种方法中均对牛肉有荧光信号显示,两种方法具有牛源性特异性,检测数值与实际样品数值基本一致,且两种方法抗干扰性较好,当存在其他肉类干扰时,仍可准确监测出牛源性,两种方法的最大误差分别为3.8%和4.0%;可定量检测不同肉制品中牛源性成分,两种方法均未检测出样品10牛肉丸中牛源性成分,验证了市面上确实存在假肉情况。说明所用方法能够准确、快速地检测出肉制品中的牛源性成分,适用于市场中肉制品的检测,对保障消费者的权益和健康具有十分重要的意义。

Detection of Bovine Components in Meat Products Based on Real-time Fluorescence Quantitative PCR and Digital PCR

In order to detect bovine-derived components in meat products quickly and effectively, real-time fluorescence quantitative PCR and digital PCR were used to detect bovine-derived components in meat products. Firstly, the specific primers and probes were designed by multiple sequence alignment according to the specific region of beef single replication genome, then the standard genomic DNA was extracted by phenol-chloroform method. Real-time fluorescence quantitative PCR and digital PCR reaction conditions were established. The purity and concentration of DNA template were determined and the extracted DNA solution was free of impurities. On this basis, the fluorescence quantitative analysis of bovine-derived components was constructed. The specificity, anti-interference and bovine-derived components in meat products were detected by standard curve of PCR. The results showed that both primers and probes have fluorescence signals for beef. The two methods have bovine origin specificity, the detection values are basically consistent with the actual sample values. The two methods have good anti-interference ability. When there are other meat disturbances, the bovine origin can still be accurately monitored. The maximum errors of the two methods were 3.8% and 4%, respectively. The bovine origin components in different meat products can be quantitatively detected. The bovine origin components in 10 beef balls of samples can not be detected by all methods, which verifies the market. Fake meat does exist. It showed that the proposed method can accurately and rapidly detect bovine-derived ingredients in meat products, and is suitable for the detection of meat products in the market. It is of great significance to protect the rights and health of consumers.