肉制品中牛源性成分荧光定量聚合酶链式反应方法的建立与应用  

Establish and Application of a Real-Time Fluorescence-Based Polymerase Chain Reaction Assay for Detection of Bovine-Derived Materials in Meat Products

作　　者：苗丽 李志娟 王珊 张秀平 陈静 

机构地区：[1]河南出入境检验检疫局检验检疫技术中心,河南郑州450003 [2]河南检验检疫鉴定咨询中心,河南郑州450003

出　　处：《肉类研究》2015年第9期30-33,共4页Meat Research

基　　金：国家质检总局科技计划项目(2014IK089)

摘　　要：为了能够快速准确检测出市场中掺假牛肉制品,采用Taq Man探针法,建立一种用于快速有效检测牛肉制品中牛源性成分的荧光定量聚合酶链式反应(polymerase chain reaction,PCR)方法。选择Gen Bank中牛β-actin基因的保守序列,设计并合成特异性引物及Taq Man探针,以构建的p MD-18T-96-beef质粒为标准品,优化反应条件。结果表明:重组质粒标准品经PCR、测序和酶切鉴定正确,建立的标准曲线Ct值与模板拷贝数对数值之间呈良好的线性关系,相关系数为0.98,斜率为-3.345,敏感度为46.1 copies/μL。特异性实验表明,用该方法检测猪肉、羊肉、鸡肉、鸭肉、鹅肉等非牛肉制品结果均为阴性。批内重复实验变异系数均小于0.98%,批间重复实验变异系数均小于0.33%,表明该方法重复性良好。用建立的荧光定量PCR方法与SN/T 2051—2008《食品、化妆品和饲料中牛羊猪源性成分检测方法实时PCR法》同时对市场上40份牛肉加工品进行检测,结果显示,本方法检测有3份样品为牛源成分阴性,与SN/T 2051—2008方法检测结果相同。可见,本实验建立的荧光定量PCR方法具有特异、敏感、快速的特点,适用于检测市场中肉类掺假的行为。A real-time fluorescence-based polymerase chain reaction（PCR） assay that enables rapid and accurate identification of adulterated beef products was developed using Taq Man probe for the rapid and effective detection of bovine-derived materials in meat products. A pair of specific primers and Taq Man probe was designed according to the conserved sequence of the bovine β-actin gene. PCR reaction conditions were optimized using p MD-18T-96-beef plasmid as standard. The results showed that the p MD-18T-96-beef plasmid was confirmed to be valid by PCR, sequencing and enzyme digestion and the developed standard curve produced a good linear relationship between Ct value and initial amounts of total DNA with correlation coefficient and slope of 0.98 and-3.345, respectively. The sensitivity was 46.1 copies/μL. No cross-reactions were found in specimens containing pork, mutton, chicken, duck and goose. The intra- and inter-assay variable coefficients were less than 0.98% and 0.33%, respectively, suggesting good repeatability. A total of 40 beef product samples from the market were measured by this method and the routine real-time PCR method from the industry standard SN/T 2051—2008. Of the 40 samples, 3 were detected as positive as consistently indicated by the two assays. This real-time PCR method with strong specificity and high sensitivity could provide a useful approach for the identification of meat adulteration.

关 键 词：肉制品 牛源性成分 荧光定量PCR 肉类掺假 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]