摘要
《中华人民共和国药典》中收载多基原药材较多,其不同来源的药材混合使用现象较为普遍,对临床用药的稳定性具有一定影响。本研究采用焦磷酸测序技术,从常用DNA条形码序列中筛选物种特异性单核苷酸多态性(single nucleotide polymorphism,SNP)位点,建立一种快速准确的辨别淫羊藿混合药材粉末中基原种的分子鉴定方法。通过多序列比对分析发现ITS序列第176位(C/T)和196位处(A/G)变异、matK序列第123位处(C/G)变异以及rbcL序列第892位处(A/C)变异可分别作为鉴别箭叶淫羊藿、朝鲜淫羊藿、淫羊藿及柔毛淫羊藿的特有SNP位点。本研究从灵敏性和稳定性两个方面考察了焦磷酸测序和Sanger测序方法在混合样品测序中的适用性。焦磷酸测序方法对混合样品中低含量样品的检测灵敏度高于Sanger测序方法。稳定性考察发现,焦磷酸测序技术对高温95℃水浴45 min后的模板DNA扩增产物仍可获得有效测序结果,而Sanger测序方法的临界点为30 min。本研究开发了一种新的基于焦磷酸测序和特异性SNP位点的淫羊藿混合粉末基原物种鉴定技术,可对淫羊藿药材的混合使用进行快速准确鉴定,灵敏性和稳定性高,也可为药材不同基原物种鉴定及混合药材粉末基原鉴定提供技术支撑,有利于保障临床用药的一致性和稳定性。
There are many multi-original medicinal materials in Chinese Pharmacopoeia,and the mixed use of medicinal materials from different sources is common,which has certain influence on the stability of clinical medication.In this study,pyrosequencing technology was used to screen species-specific single nucleotide polymorphisms(SNP)from commonly used DNA barcode sequences,and a rapid and accurate molecular identification method for original species in mixed medicinal powder of Epimedii Folium was established.Multiple sequence alignment analysis showed that the 176th(C/T)mutation and the 196th(A/G)mutation of ITS,the 123rd(C/G)mutation of matK and the 892nd(A/C)mutation of rbcL could be used as the unique SNPs of E.sagittatum,E.koreanum,E.brevicornu and E.pubescens,respectively.In this study,the applicability of pyrosequencing and Sanger sequencing methods in the sequencing of mixture samples was investigated from the perspective of sensitivity and stability.Pyrosequencing method has higher detection sensitivity than Sanger sequencing method for low content samples in the mixed samples.Stability analysis showed that pyrosequencing technology could still obtain effective sequencing results for the amplified products of template DNA after 45 min of 95℃high temperature water bath,while the critical point of Sanger sequencing method was 30 min.In this study,a new identification technology of Epimedii Folium mixed powder primordial species based on pyrosequencing and specific SNP was developed,which can quickly and accurately identify the mixed use of Epimedii Folium with high sensitivity and stability,and can also support the identification of different primordial species and mixed powder primordial herbs,which is conducive to ensuring the consistency and stability of clinical medication.
作者
刘姿怡
裴艺菲
冯雪
赵贵萍
陈铁柱
李西文
LIU Zi-yi;PEI Yi-fei;FENG Xue;ZHAO Gui-ping;CHEN Tie-zhu;LI Xi-wen(Department of Pharmacy,Southwest Medical University,Luzhou 646000,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Sichuan Provincial Key Laboratory of Quality and Innovation Research of Chinese Materia Medica,Sichuan Academy of Traditional Chinese Medicine Sciences,Chengdu 610041,China)
出处
《药学学报》
CAS
CSCD
北大核心
2023年第8期2532-2540,共9页
Acta Pharmaceutica Sinica
基金
中国中医科学院科技创新工程项目(CI2021A03910,CI2021A04106)
中央级公益性科研院所基本科研业务费专项资金(ZZ15-YQ-033,ZXKT21026,ZXKT23004)
四川省科技厅科技创新人才(2021JDRC0043)
国家中医药管理局舒光明全国名老中医药专家传承工作室项目(2022)。
