摘要
目的:建立大籽蒿与其混淆品细叶亚菊特异性DNA荧光可视化鉴别方法。方法:将大籽蒿与细叶亚菊的内转录间隔区(ITS)、rbcL、psbA-trnH序列进行比对,构建系统发育树,选择最佳DNA条形码,寻找差异位点,设计大籽蒿与细叶亚菊的特异性鉴别引物,建立特异性DNA快速检测体系。将扩增体系与SYBR GreenⅠ荧光染料相结合,实现荧光可视化检测。利用建立的位点特异性聚合酶链式反应(AS-PCR)荧光可视化鉴别方法,对3批五味甘露药浴汤散中原料药进行鉴别。结果:ITS为大籽蒿与细叶亚菊最佳的品种鉴定序列;基于ITS建立AS-PCR荧光可视化鉴别方法可在40 min内完成特异性检测。采用该方法对3批五味甘露药浴汤散进行分析,3批样品均检测出大籽蒿,但其中1批样品混有细叶亚菊。结论:建立的方法特异性良好,可以对大籽蒿及其藏成药中原料药进行快速、准确的可视化鉴别。
Objective:To develop a species-specific DNA fluorescence visualization method for distinguishing Artemisia sieversiana from its adulterant Ajania tenuifolia.Methods:The internal transcribed spacer(ITS),rbcL,and psbAtrnH sequences of Artemisia sieversiana and Ajania tenuifolia were compared.A phylogenetic tree was constructed,and the optimal DNA barcode was selected.Based on the differential sites,specific identification primers for Artemisia sieversiana and Ajania tenuifolia were designed to establish a specific DNA rapid detection system.The amplification system was combined with SYBR GreenⅠfluorescent dye to achieve fluorescence visualization detection.The established allelespecific polymerase chain reaction(AS-PCR)fluorescence visualization method was used to identify the raw materials in three batches of Wuwei Ganlu Yaoyu Tangsan.Results:ITS was selected as the optimal identification sequence for distinguishing Artemisia sieversiana from Ajania tenuifolia.The AS-PCR fluorescence visualization method based on ITS enabled specific detection within 40 minutes.By using this method to analyze three batches of Wuwei Ganlu Yaoyu Tangsan,Artemisia sieversiana was detected in all three batches,while one batch was found to be adulterated with Ajania tenuifolia.Conclusion:The established method demonstrates good specificity and can be used for the rapid and accurate visualization identification of Artemisia sieversiana and its raw materials in Tibetan medicine preparations.
作者
谢雨宸
王亚倩
李慧林
赵程成
陈蓉
范刚
XIE Yuchen;WANG Yaqian;LI Huilin;ZHAO Chengcheng;CHEN Rong;FAN Gang(School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China;Jiangyou People's Hospital,Jiangyou 621700,China;School of Ethnic Medicine,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China)
出处
《中国现代中药》
2025年第7期1238-1244,共7页
Modern Chinese Medicine
基金
国家重点研发计划项目(2023YFC3504403)。
关键词
大籽蒿
细叶亚菊
位点特异性聚合酶链式反应
荧光可视化
藏成药
Artemisia sieversiana Ehrhart ex Willd.
Ajania tenuifolia(Jacq.)Tzvel.
AS-PCR
fluorescence visualization
Tibetan medicine preparation
