实时荧光定量聚合酶链反应技术检测食品中动物源性成分单拷贝核基因靶标

Determination of single-copy nuclear gene targets of animal-derived components in food by real-time fluorescence quantitative polymerase chain reaction technology

中文关键词:  动物源性食品  实时荧光定量PCR  核基因

英文关键词:animal-derived food  real-time fluorescence quantitative polymerase chain reaction  nuclear gene

中文摘要:

      目的 建立针对食品中动物源性成分单拷贝核基因靶标的实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction, qPCR)检测方法。方法 以食品中常见动物源性成分特异性核基因为靶标设计qPCR引物及Taqman探针, 验证及确认方法的特异性、灵敏度, 采用模拟及实际市售动物源性食品检测方法的适用性及稳定性, 并同胞质线粒体基因靶标qPCR扩增检测值比较分析。结果 单拷贝核基因靶标的qPCR引物探针特异性良好, 其最低检出限可达0.1 ng/μL DNA质量浓度, 核基因靶标能够准确识别0.10%(质量比)的最低掺假成分含量; 核基因靶标扩增反应的扩增循环数(cycle threshold, Ct)值大于胞质靶基因Ct值, 胞质靶基因可识别更低0.01%(质量比)的掺假含量; 在对模拟及实际市售样品检测中, 核基因qPCR反应可以准确、有效检测出掺有不同源性成分的动物源样品, 且不易检出鸡精配料及生产线其他源性污染成分。结论 本研究结果表明基于单拷贝核基因靶标建立的qPCR扩增体系对食品中常见动物源性成分检测的特异性强, 灵敏度高, 同胞质靶基因扩增结果相比, 其检测结果不易受配料代入和加工过程中交叉污染的影响, 可快速准确检测动物源性成分掺假情况, 利于市场监管及风险研判。

英文摘要:

      Objective To establish a method for determination of single-copy nuclear gene targets of animal-derived components in food by real-time fluorescence quantitative polymerase chain reaction (qPCR). Methods qPCR primers and Taqman probes were designed to target the specific nuclear genes of common animal-derived components in food to verify and confirm the specificity and sensitivity of the method. The applicability and stability of simulated and actual commercially available animal-derived food detection methods were adopted, and the qPCR detection values were compared and analyzed with those of mitochondrial cytoplasmic gene target detection. Results The qPCR primer probe for single-copy nuclear gene targets had good specificity, with a minimum limit of detection of 0.1 ng/μL DNA mass concentration, nuclear genes could accurately identify the minimum adulteration component content of 0.1% (mass ratio). The cycle threshold (Ct) value of the nuclear genes amplification reactions were all greater than that of the cytoplasmic target gene, and the amplification reactions of cytoplasmic genes could recognize a lower adulteration content of 0.01% (mass ratio). In the testing of simulated and actual commercially available samples, the nuclear gene qPCR reaction could accurately and effectively detect animal-derived samples mixed with different source components, and it was not easy to detect chicken essence ingredients and other source contamination components from the production line. Conclusion The research results of this paper show that the qPCR amplification system established based on single-copy nuclear gene targets has strong specificity and high sensitivity for the detection of common animal-derived components in food. Compared with the amplification results of mitochondria genes, the nuclear genes’ detection method is less affected by the substitution of ingredients and the cross-contamination on the production line, and can quickly and accurately detect animal-derived component in food, which make it conducive to market supervision and risk assessment.