基于实时荧光PCR定量检测肉制品猪源性成分方法的建立  

Establishment of a Method to Quantitatively Detect Pork-Derived Ingredients in Meat Products Based on Real-Time PCR

作　　者：蒲长宇 安得霞 王戊腾 陈妍[1] 卢建雄[1] 张国华[1] Pu Changyu;An Dexia;Wang Wuteng;Chen Yan;Lu Jianxiong;Zhang Guohua(College of Life Science and Engineering, Northwest University for Nationalities, Lanzhou, 73003)

机构地区：[1]西北民族大学生命科学与工程学院,兰州730030

出　　处：《基因组学与应用生物学》2017年第11期4585-4591,共7页Genomics and Applied Biology

基　　金：西北民族大学生命科学众创空间扶持项目;"动物营养与饲料科学教学团队"建设项目;国家自然科学基金(No.31460589;No.31560639)项目基金共同资助

摘　　要：为了建立肉制品中猪源性成分检测的PCR方法,以猪线粒体细胞色素b(cytochrome b,Cyt b)为目的基因,设计猪特异性PCR引物;以猪、牛、羊、鸡及鸭DNA和不同稀释浓度猪DNA进行Real-time PCR反应,证明了该引物扩增具有物种特异性,检测低限可达5×10-5 ng/μL;设计内参基因β-actin通用引物,以猪源性成分含量0%~100%的混合肉样DNA为模板扩增,建立标准曲线,2-△△Ct值与猪源性成分含量有良好线性关系(R2=0.991 4);对猪源性成分含量0%~75%的混合肉样检测,回收率为99.85%~102.60%。应用建立的PCR方法检测了市售的6种品牌火腿肠,标识为清真食品的5种均未检测到猪源性成分,未标识清真食品的猪源性成分含量为12.70%。建立的实时荧光PCR检测方法操作简便、特异性强、所得数据可靠,为肉制品猪源性成分检测提供了新的手段。In order to build a PCR method to determinate pig derivatives in meat products, the pig specific PCR primer was designed with the pig mitochondrial cytochrome b （cytochrome b, Cyt b） as the target gene; The genome DNA of pig, cattle, sheep, chickens and duck were used to conduct PCR Real-time reaction with pig DNA of differ- ent concentration. The results showed that this primer was amplified with species specificity and the minimum detectability could reach 5 ×10^-5 ng/μL. Took reference gene β-actin as the universal primer and the DNA of mixed meat which contained 0%-100% of pork-derived ingredients as template to amplify and constructed the standard curve. From the curve we obtained that the value of 2^-△△a and the content of pig derivatives had a good linear relationship （R2=0.991 4）. The mixed meat which contained 0%-75% pork-derived ingredients were detected by the standard curve, and the coefficient of recovery was 99.85%-102.60%. Six kinds of brand sterilixed sausages from the market were detected by using the established PCR method. No pork-derived ingredients was found in five brands marked halal food, and 12.7% pork-derived ingredients was detected in one brand unmarked halal food. The real-time fluorescent PCR detection method established by this study was easy, reliable and with high specificity, which provided a new way to detect pork-derived ingredients in meat products.

关 键 词：肉制品 猪源性成分 实时PCR 定量检测 

分 类 号：Q78[生物学—分子生物学] TS251.7[轻工技术与工程—农产品加工及贮藏工程]