荧光定量PCR方法鉴别肉制品中羊源性成分

Real- time PCR detection of sheep derived materials in meat product

根据羊线粒体DNA细胞色素b基因序列,设计特异性引物和Taqman探针。通过反应条件的优化筛选,构建标准曲线,建立灵敏、可靠的肉制品中羊源性成分的实时荧光PCR鉴别方法。结果表明,所设计的引物可有效地进行肉制品中羊肉成分的鉴别,特异性强,灵敏度高达16pg/μL;标准曲线扩增效率达98.561%,R2=1,符合《实时荧光定量国际化标准-MIQE指南》要求;本研究建立的方法检测结果与国标GB/T 20190-2006方法结果符合率达100%,且不需电泳、酶切和测序等步骤。通过对市售肉制品样本的鉴别验证了方法的实用价值,为肉制品质量的有效控制提供了新的途径。

Targeting cytochrome b gene of mitochondrial were designed.After optimization of reaction conditions DNA from sheep,the specific primers and TaqMan probes and establishment of standard curve, a fluorescent real- time PCR method had been set up to detect sheep derived materials.The results showed that the specific primers could identify sheep derived materials in meat product specially and effectively,and the sensitivity was as high as 16pg/μL.Amplification efficiency of the standard curve was 98.561% and correlation coefficient R2 was 1.000.These properties meet the requirement of the《MIQE Guide》.The results obtained according to the new method and the National Standard （ GB/T20190Y-2006 routine PCR method） were exactly the same, and electrophoresis, restriction digestion and sequencing were avoided in the new method. The value of this method was proved though the identification of the meat product from the market.The assay provides a new way for the quality control of the meat products.