实时荧光PCR定量检测肉制品中猪源性成分

为了准确可靠地对肉制品中猪源性成分进行定量检测，通过生物信息学方法筛选到猪细胞核单拷贝基因（carcinoembryonic antigen-related cell adhesion molecule 2-like，CACA）。以CACA基因为扩增靶标，设计了特异性引物、TaqMan探针，建立了基于实时荧光定量PCR技术检测猪肉含量的方法。实验结果表明，该方法具有良好的种外特异性和种内稳定性，在20~0.032 ng/μL的DNA浓度范围内，模板DNA和扩增Ct值之间存在良好的线性关系，标准曲线为y=?3.508x+28.636，线性决定系数R2为0.997。在相对标准偏差≤25%的条件下，可准确检测猪DNA含量低至0.1%的DNA样品，猪肉含量低至5.0%的肉制品。通过对市售样品的检测，发现有的肉制品中掺有猪肉但未标识，有的配料表中猪肉并非主要成分但实际上猪肉占主要成分。本研究建立的实时荧光定量PCR能够准确可靠对肉制品中的猪肉成分进行检测，对准确鉴别肉制品掺假以及量化肉制品组分具有重要参考价值。

Detection of Pork-Derived Ingredients Based on Real-time Quantitative PCR

In order to accurately and reliably quantify pork-derived ingredients in meat products, single-copy gene (carcinoembryonic antigen-related cell adhesion molecule 2-like, CACA) in pig cell nucleus was screened by bioinformatics methods, with CACA gene as the amplification target, specific primers and TaqMan probes were designed, and a TaqMan real-time quantitative PCR method for detecting pork ingredients was established. The experimental results showed that the method had good extraspecies specificity and intraspecies stability. There was a good linear relationship between the template DNA and the amplified Ct value within the DNA concentration range from 20 to 0.032 ng/μL. The standard curve was y=?3.508x+28.636, the coefficient of determination (R2) value was 0.997. Under the condition of relative standard deviation ≤25%, it could accurately detect DNA samples with pork DNA content as low as 0.1%, and meat products with pork content as low as 5.0%. Through testing of commercially available samples, it was found that somemeat products were mixed with pork but were not labeled. And some meat products, pork was not listed as the main ingredient but in fact pork was the main ingredient. The real-time quantitative PCR method established in this study could be used to accurately and reliably detect pork ingredients in meat products. It has important reference value for accurately identifying adulteration of meat products and quantifying the ingredients of meat products.