基于超快速VPCR荧光可视化检测技术的小檗皮及其藏成药中原料药的真伪鉴别研究

Identification of Berberis Cortex and its Tibetan patent medicine based on Ultra-fast VPCR and fluorescence visual identification technology

目的建立藏药小檗皮BerberisCortex及藏成药中小檗皮特异性DNA的超快速荧光可视化检测方法,实现对小檗皮药材及藏成药中小檗皮的快速鉴别。方法将小檗皮及其混淆品的ITS2序列进行比对,筛选出小檗皮药材特异性DNA序列;针对该序列设计小檗皮特异性引物,建立小檗皮药材以及藏成药中小檗皮特异性DNA的超快速VPCR扩增体系;后将该扩增体系与SYBR Green I荧光染料相结合,实现对扩增产物的荧光可视化检测;同时对该体系的特异性、灵敏度以及在真实样本中的适用性进行考察。结果该体系可以在40min内完成对小檗皮以及藏成药中小檗皮特异性DNA的扩增及荧光可视化检测;检测限低至10pg/μL;对16个批次的小檗皮药材进行分析,其中15个批次的样本产生阳性信号鉴定为小檗皮,与测序结果100%一致,1个批次的样本产生阴性信号,该样本经通用引物测序鉴定为黄柏;对3种含有小檗皮的藏成药进行分析,3个样本均产生阳性信号。结论所建方法缩短了检测时间,并且特异性良好,灵敏度高,可以对小檗皮及其藏成药中小檗皮进行快速、准确的可视化鉴别。

Objective To develop an ultra-fast VPCR fluorescence visual identification method for Xiaobopi(Berberis Cortex)-specific DNA to realize fast identification of Berberis Cortex and its Tibetan patent medicine.Methods Specific primers for Berberis Cortex were designed according to the alignment results of the Berberis Cortex and its confusions.A fast VPCR amplification system was established for Berberis Cortex-specific DNA both in crude drugs and in Tibetan patent medicine.Moreover,this fast VPCR assay is also developed as a visual detection method by combing SYBR Green I fluorescent dye.Characters of an identification method including specificity,sensitivity,and generality were systematically studied.Results The amplification and fluorescence visual identification of Berberis Cortex-specific DNA could be finished within 40 minutes.The detection limit is as low as 10 pg/μL.A total of 16 batches of Berberis Cortex were analyzed by the developed method,of which 15 batches generated positive signals and one batch generated negative signal which was identified as Huangbo(Phellodendri Chinensis Cortex)using DNA sequencing method.All these results were 100%consistent with the sequencing results.Three kinds of Tibetan patent medicines containing Berberis Cortex were analyzed and all samples yielded positive signals.Conclusion The developed method not only greatly shortens the detection time,but also has a good specificity and sensitivity,which can effectively identify Berberis Cortex and its Tibetan patent medicine.