Taqman探针荧光PCR法检测熊源性成分

Detection of Ursus-Derived Ingredients by TaqMan Probe Fluorescent PCR

为了对熊源性成分进行有效鉴定,本研究根据熊科动物线粒体DNA(mtDNA)序列,使用分子生物学软件Primer Express 3.0设计了一套特异性引物、探针,用以建立Taqman探针荧光PCR检测熊源性成分的方法。结果显示,所建立方法特异性强,15份熊属动物组织样品均出现特异性扩增曲线,20份阴性对照动物样品均未出现扩增曲线;方法灵敏度高,最低可以检测到10个拷贝数量级的重组质粒DNA,对熊胆粉稀释106倍后,仍可扩增出熊DNA;方法稳定性好,对相同的样品在不同时间进行2次重复检测,批内变异系数分别为0.88%、1.13%,批间变异系数为1.38%。结果表明,本研究建立的方法可应用于熊属动物组织及其加工品中的熊源性成分的检测。

To establish the TaqMan fluorescent PCR method for detecting Ursus-derived ingredients,specific primers and a Taqman probe were designed using software Primer Express 3. 0. The sequence of the primers and probe were based on mitochondrial DNA sequence of Ursidae. Our results indicated high specificity,high sensitivity and high stability. The specific amplification curve only occurred in 15 ursine samples, while all of 20 negative samples showed no amplification curve. The minimum detectable amount of recombinant plasmid DNA was 10 copies,and the DNA in the Ursus thibetanus bile powder could be amplified even if it was diluted 106 times. The intra-assay variation coefficients were 0. 88% and 1. 13%, and the inter-assay variation coefficient was 1. 38% in a duplicate assay of the same samples at different times. The method established in this research can be applied in detecting Ursus-derived ingredients in tissues and related processed products of Ursus.