一种基于多重实时荧光聚合酶链式反应熔解曲线分析的肉及-食品科学.pdfVIP

※安全检测 食品科学 2017, Vol.38, No.12 217 一种基于多重实时荧光聚合酶链式反应 熔解曲线分析的肉及肉制品掺假鉴别方法 * * 周 彤，李家鹏 ，李金春，乔晓玲 ，黄 鑫，陈 曦，杨君娜，郭 雅 （中国肉类食品综合研究中心肉类加工技术北京市重点实验室，北京 100068 ） 摘  要：为实现肉及肉制品掺假快速鉴别，分别以猪、牛线粒体DNA 的COX Ⅰ，绵羊、山羊、狗、狐狸、貉线粒 体DNA 的16S rRNA及鸡、鸭线粒体DNA 的12S rRNA基因为靶位点，设计扩增产物熔解温度（Tm值）具有显著性差 异的特异性引物，建立一种用于快速鉴别肉或肉制品中猪、牛、绵羊、山羊、鸡、鸭、狗、狐、貉9 种源性成分的 5 重实时荧光聚合酶链式反应熔解曲线分析方法，通过特异性、灵敏度及市售样品的检测，对该方法进行检验和评 价。结果表明：方法具有良好的特异性及灵敏度，单物种DNA检出限为0.001～1 ng，多物种混合DNA检出限均为 0.1 ng ，通过市售样品检测表明该方法可用于实际样品（包括生鲜样品和熟制样品）掺假的快速鉴别。 关键词：肉类掺假；多重实时荧光聚合酶链式反应；熔解曲线分析；SYBR Green染料；动物源性成分 A Multiplex RT-PCR Melting Curve Analysis Method for Adulteration Identification in Meat and Meat Products * * ZHOU Tong, LI Jiapeng , LI Jinchun, QIAO Xiaoling , HUANG Xin, CHEN Xi, YANG Junna, GUO Ya (Beijing Key Laboratory of Meat Processing Technology, China Meat Food Research Center, Beijing 100068, China) Abstract: This study was aimed to establish a rapid method for the identification of adulterated meat and meat products. Five pairs of species-specific primers, yielding amplicons having significantly different melting temperatures (Tm), were designed targeting the mitochondrial COX Ⅰ gene of pig and bovine, the 16S rRNA gene of sheep, goat, dog, fox and raccoon, and the 12S rRNA gene of chicken and duck, respectively. The established multiplex RT-PCR-melting curve analysis method was evaluated in term of specificity and sensitivity and was applied in the detection of commercial meat products. The method showed a good specificity and sensitivity, and was able to detect 0.001–1 ng of DNA from single species and 0.1 ng of DNA