含有扩增内标的食品中猪肉和鸡肉成分Taqman探针实时荧光PCR检测方法的建立

【目的】建立含有扩增内标（IAC，Internal amplification control）的用于食品中猪肉和鸡肉成分鉴别的Taqman探针实时荧光PCR检测方法。【方法】分别基于猪的beta actin 基因和鸡的transforming growth factor 基因设计引物和探针；考察引物和探针的特异性与灵敏度；设计并构建扩增内标，优化体系中扩增内标浓度，建立内标实时荧光PCR体系；使用该检测体系对鲜肉和熟肉来源的DNA模板进行检测评价；应用该体系对市售38份样本进行盲样检测。【结果】含有扩增内标的Taqman探针实时荧光PCR体系能有效地进行食品中猪肉和鸡肉成分的快速检测，非目标物种在40个循环内均无出现扩增，检出限均为0.5 ng DNA；该体系对鲜肉和熟肉来源的DNA模板检测无显著差异；对市售食品样本的鉴别验证了方法的实用价值。【结论】该方法准确稳定，可用于食品中猪肉和鸡肉成分的快速检测。

Establishment of a Taqman Real-time PCR with Internal Amplification Control for the Detection of Chicken and Pork Ingredient in Food Products

【Objective】A Taqman real-time PCR with IAC (Internal amplification control) was developed to identify pork and chicken ingredient in meat products. 【Method】 Specific primers and probes were designed based on the sequence of beta actin gene of pig DNA and transforming growth factor gene of chicken DNA, respectively. The specificity and sensitivity of the primers and probes were evaluated. An IAC was designed and constructed, and the real-time PCR system with IAC was established by optimizing the concentration of IAC in the system. The DNA of raw and cooked samples was evaluated by applying the Taqman real-time PCR system with IAC. Meanwhile, 38 food samples from market were assayed. 【Result】The pork and chicken ingredient in food was detected effectively by the Taqman real-time PCR with IAC. No cross-reaction was observed between species specific primer-probe systems in 40 cycles. The limit of detection was 0.5 ng of template DNA. No significant difference was observed between the Ct values of raw and cooked samples. The accuracy of the method was verified by examination of 38 food samples from the market.【Conclusion】An accurate and reliable method was developed to identify pork and chicken ingredient in food products.