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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。
In order to validate the Xiong-Yuan sex composition, this study designed a set of specific primers and probes to establish Taqman probe fluorescent PCR based on mitochondrial DNA (mtDNA) sequence of the Bears Division using the molecular biology software Primer Express 3.0 Method of detecting bear derived components. The results showed that the established method was specific and specific, and all of the 15 Ursus animal tissues showed specific amplification curves, and none of the 20 negative control animals showed any amplification curve. The method was characterized by high sensitivity and low detection limit of 10 copies Of the recombinant plasmid DNA, the Xiong Dan powder diluted 106 times, still can amplify the bear DNA; method stability is good, the same sample at different times for 2 repeated testing, within the batch coefficient of variation were 0.88%, 1.13 %, Coefficient of variation between batches was 1.38%. The results show that the method established in this study can be applied to the detection of bear-derived components in the animal tissues and processed products of Ursus.