双重实时荧光PCR法检测食品和饲料中的鸡源性成分

Duplex Fluorescent Real-time PCR Detection of Chicken Derived Materials in Food and Feed

根据鸡线粒体DNA细胞色素b基因序列和内参照基因PUC18质粒基因序列设计特异性引物和以不同荧光素标记的TaqMan探针。通过对反应条件的优化筛选,建立能同时扩增鸡源性成分和内参照基因成分的双重实时荧光PCR检测方法。设置内参照反应是为了监控反应体系中是否含有PCR反应的抑制物,避免出现假阴性结果。分别以鸡、鸭、鹅、火鸡、牛、羊、猪、鱼、兔、驴、鹿、狗、马、鸽子、大豆、玉米、小麦、大米、马铃薯及番茄的线粒体DNA作为模板进行特异性试验,结果表明该方法仅能特异性扩增鸡源性成分,而对其它物种未见有效扩增。通过灵敏度测试,该方法检出限达0.01%。所制定的方法特异性高,灵敏度好,可以作为食品和饲料中鸡源性成分的高效检测方法。

We designed the specific primers and TaqMan probes targeting cytochrome b genes of chicken mitochondrial DNA and PUC 18 plasmid genes for the internal positive control （ IPC ） . We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a duplex fluorescent real-time PCR method to detect chicken derived materials and IPC, simuhaneously. In order to detect inhibition, IPC were used in all PCR reactions, which prevented false negative results. The specificity of the method was evaluated using template DNAs from chicken and other 19 species such as turkey, duck, goose, bovine, ovine, pork, fish, rabbit, donkey, venison, dog, horse, pigeon, soybean, maize, wheat, rice, potato, tomato,only the specific amplification of chicken derived materials was observed,where; as no amplification products were obtained from other species. The limit of quantification was found to be 0.01% through sensitivity tests. In conclusion, the study showed that the developed real-time PCR method is a specific, sensitive and efficacious assay for the detection of chicken derived materials in food and feed.