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目的建立一种快速、简便、准确的分子生物学方法,用来鉴别鹿鞭真伪及品种。方法应用柱色谱法提取梅花鹿鞭、马鹿鞭、牛鞭以及市售鹿鞭线粒体DNA;根据GenBank中鹿类动物线粒体细胞色素b基因序列,设计一对引物用于线粒体DNA的聚合酶链式反应(PCR)特异性扩增;应用变性聚丙烯酰胺凝胶电泳对聚合酶链式反应产物进行单链构象多态分析(single strand conformation polymorphism,SSCP)。结果梅花鹿鞭、马鹿鞭以及部分市售鹿鞭可显示清晰且迁移率不同的片段;对照组伪品鹿鞭及部分市售鹿鞭无特异性显示。结论柱色谱法提取线粒体DNA所需时间短,DNA纯度高,DNA分子的破坏性较小,为特异性聚合酶链式反应扩增提供一级结构保证;单链构象多态分析法可以清晰显示正品梅花鹿鞭、马鹿鞭以及部分市售鹿鞭单链片段,其一级结构中的碱基差别为正品梅花鹿鞭、马鹿鞭的不同迁移率提供依据,也为药源市场鹿鞭的鉴别提供一种可以直接进行亚种间鉴别的快速、简便、准确的可行性方法。
Objective To establish a rapid, simple and accurate molecular biology method to identify the authenticity and variety of deer whip. Methods The mitochondrial DNA of Sika deer whip, Cervus nippon, bullwhip and commercially available Dew whip was extracted by column chromatography. According to the mitochondrial cytochrome b gene sequence of deer in GenBank, a pair of primers was designed for polymerase chain reaction of mitochondrial DNA (PCR). The single strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR) products was performed by denaturing polyacrylamide gel electrophoresis. Results Sika deer whip, red deer whip and some commercially available deer whip can show a clear and different mobility fragments; control group of counterfeit deer whip and some commercially available deer whip no specific display. Conclusion The time required for the extraction of mitochondrial DNA by column chromatography is short, DNA purity is high and DNA molecule is less destructive, which provides a first-order structure guarantee for specific polymerase chain reaction amplification. Single-strand conformational polymorphism analysis can clearly show Genuine Sika deer whip, red deer whip and some commercially available single-stranded fragments of deer whip, the primary structure of the base difference for the authentic Sika deer whip, red deer whip different migration basis, but also for the identification of drug source market Lubian It provides a fast, easy and accurate feasible method that can directly identify subspecies.