同时检测速冻食品中鸡、鸭、猪和牛源性成分实时荧光聚合酶链式反应方法的建立及应用

以鸡转化生长因子β-3(transforming growth factor beta-3,TGFB3)基因、猪朊蛋白(prion protein,PRNP)基因和鸭、牛生长激素(growth hormone,GH)基因为靶基因,设计合成特异性引物和TaqMan探针,通过对实时荧光聚合酶链式反应(polymerase chain reaction,PCR)体系和反应条件的优化,建立同时检测速冻食品中鸡、鸭、猪和牛源性成分的实时荧光PCR检测方法。结果表明:本研究所建立的方法只在鸡、鸭、猪或牛源性成分存在时才产生特异性扩增曲线,表明该方法具有良好的特异性;且对鸡、鸭、猪和牛源性成分的最低检测质量浓度分别可达到10.0、1.0、10.0、10.0 pg/μL,具有良好的灵敏性;应用建立的检测方法对市售34份速冻食品鸡、鸭、猪和牛源性成分进行同时检测,能够实现对速冻食品中4种动物源性成分的有效检测,进一步分析发现,15份速冻食品源性成分测定结果与外包装标注成分不一致。本研究所建立的同时检测速冻食品中鸡、鸭、猪和牛源性成分的实时荧光PCR检测方法具有良好的特异性和灵敏性,且操作简单、适用范围广,能够满足日常检测需求。

Development and Application of a Real-Time Polymerase Chain Reaction Assay for Simultaneous Detection of Chicken-, Duck-, Swine- and Bovine-Derived Ingredients in Quick-Frozen Food

A TaqMan real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of chicken-, duck-, swine- and bovine-derived ingredients in quick-frozen food. Specific primers and TaqMan probes were designed and synthesized using the transforming growth factor beta-3 (TGFB3) gene of chicken, the prion protein (PRNP) gene of swine and the growth hormone (GH) gene of duck and bovine as target genes. The PCR reaction systems and conditions were optimized. The developed real-time PCR assay demonstrated good specificity, in which specific amplifications were observed for chicken, duck, swine and bovine. The assay also showed high sensitivity with limits of detection for chicken-, duck-, swine- and bovine-derived ingredients of 10.0, 1.0, 10.0 and 10.0 pg/μL, respectively. The PCR assay could detect the four kinds of animal-derived ingredient in 34 commercial quick-frozen foods. Further analysis revealed that the results of the PCR assay for 15 quick-frozen foods were inconsistent with the ingredients on the label. The real-time PCR assay developed in this study has good specificity and sensitivity, and it is simple to operate and suitable for the rapid detection of animal-derived ingredients in quick-frozen foods.