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

摘要

A real-time fluorescence-based polymerase chain reaction (PCR) assay that enables rapid and accurate identification of adulterated beef products was developed usingTaqMan probe for the rapid and effective detection of bovine-derived materials in meat products. A pair of specific primers andTaqMan probe was designed according to the conserved sequence of the bovine β-actingene. PCR reaction conditions were optimized using pMD-18T-96-beef plasmid as standard. The results showed that the pMD-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 betweenCt 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.%为了能够快速准确检测出市场中掺假牛肉制品，采用TaqMan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GenBank中牛β-actin基因的保守序列，设计并合成特异性引物及TaqMan探针，以构建的pMD-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。