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

摘要

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 COXⅠ 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 form mixed species.Thus,it can be used to test both raw and processed meat products.%为实现肉及肉制品掺假快速鉴别,分别以猪、牛线粒体DNA的COXⅠ,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.