肉制品中鸭源性成分TaqMan探针实时荧光PCR检测方法的建立与应用

Development and application of a TaqMan real-time PCR assay for rapid detection of duck-derived ingredients in meat products

中文关键词:  鸭源性成分  生长激素基因  实时荧光PCR  快速检测

英文关键词:duck-derived ingredients  growth hormone gene  real-time PCR  rapid detection

中文摘要:

      目的 建立TaqMan探针实时荧光PCR法快速检测肉制品中鸭源性成分的分析方法。方法 以鸭生长激素(growth hormone, GH)基因为靶基因, 基于特异性保守序列设计引物和TaqMan探针, 优化反应体系和反应条件, 建立了鸭源性成分实时荧光PCR(real-time PCR)检测方法。结果 所建立的real-time PCR方法特异性强, 仅对鸭基因组DNA出现特异性扩增曲线, 对牛、羊、猪等其他异源性动物基因组DNA均无扩增曲线; 灵敏性高, 以鸭基因组DNA为模板, 检出限为1.0 pg; 重复性好, 不同浓度基因组DNA测定的Ct值变异系数小于4%。使用建立的real-time PCR方法对200份市售肉制品进行检测, 在11份肉制品中检出鸭源性成分, 同现行行业标准SN/T 3731.5—2013检测结果一致。结论 本研究所建立的real-time PCR方法检测具有特异性强、敏感性高、快速高效等特点, 适用于肉制品中鸭源性成分快速检测。

英文摘要:

      Objective To establish a rapid detection method for duck-derived ingredients in meat products by a TaqMan real-time PCR assay. Methods Duck growth hormone (GH) gene was used as the target gene, and a real-time PCR method for the detection of duck-derived ingredients was established by designing primers and TaqMan probes based on specific conservative sequences and optimizing the reaction system and reaction conditions. Results The established real-time PCR method had strong specificity, only showed a specific amplification curve for duck genomic DNA, and had no amplification curve for genomic DNA of other heterologous animals such as cattle, sheep, pig, and so on. It had high sensitivity, using duck genomic DNA as template, and the detection limit was 1.0 pg; the repeatability was good, and the coefficient of variation of Ct value determined by different concentrations of genomic DNA was less than 4%. A total of 200 commercial meat products were detected using the established real-time PCR method, and duck-derived ingredients were detected in 11 meat products, which were consistent with the detection results in the current industry standard SN/T 3731.5—2013. Conclusion The real-time PCR method established in this study has the characteristics of strong specificity, high sensitivity, rapidity and high efficiency, and is suitable for rapid detection of duck-derived ingredients in meat products.