应用DNA提取改良和二次聚合酶链反应技术检测乌头及其炮制品  

Detection of Aconitum Materials and Its Processed Products Using Improved DNA Extraction and Two-time PCR

作　　者：朱高倩 李双良 马莉 周培军 蒲星宇 王丽 符德欢 ZHU Gao-Qian;LI Shuang-Liang;MA Li;ZHOU Pei-Jun;PU Xing-Yu;WANG Li;FU De-Huan(Yunnan Institute of Materia Medica,Kunming 650111 Yunnan,China;Yunnan Bai Yao Group Innovation,Research and Development Center,Kunming 650111 Yunnan,China;Yunnan Provincial Company Key Laboratory for Traditional Chinese Medicine and Ethnic Drug of New Drug Creation,Kunming 650111 Yunnan,China)

机构地区：[1]云南省药物研究所,云南昆明650111 [2]云南白药集团创新研发中心,云南昆明650111 [3]云南省中药和民族药新药创制企业重点实验室,云南昆明650111

出　　处：《广州中医药大学学报》2021年第2期385-391,共7页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：云南省应用基础研究计划项目(编号:2017FD234);云南省重大科技专项(农业)(编号:2017AB005)。

摘　　要：【目的】筛选适用于9种乌头属植物根茎炮制前后DNA提取的方法。【方法】采用基于十六烷基三甲基溴化铵(CTAB)法、十二烷基硫酸钠(SDS)法的12种改良方法提取9种乌头属植物根茎炮制前后DNA,考察提取缓冲液种类、聚乙烯吡咯烷酮(PVP)添加、水浴时间、DNA沉降方式对乌头属植物根茎炮制前后DNA提取的影响,以及二次聚合酶链反应(PCR)技术对乌头炮制品ITS2片段浓度提高的影响。【结果】不同缓冲液与水浴时间互作提取DNA效应不同,2×CTAB(含2.5 mol/L NaCl)水浴3 h提取的9种乌头生品DNA的ITS2扩增效果最佳,2×CTAB(含1.4 mol/L NaCl)水浴3 h提取的9种乌头炮制品DNA的ITS2扩增效果最佳。添加PVP或添加PVP+改变DNA沉降方式对生品DNA提取影响不大,但对炮制品有明显的抑制作用。经过二次PCR可显著提高炮制品ITS2的扩增效率。【结论】提取缓冲液和水浴时间的有效互作提取DNA以及二次PCR技术的应用可提高乌头属植物根茎ITS2片段制备效率,为DNA条形码技术广泛应用于乌头属药材提供必要的技术支持。Objective To establish a method for extracting DNA from the rhizomes of 9 kinds of Aconitum plants before and after preparing. Methods Twelve modified methods based on cetyl trimethyl ammonium bromide(CTAB)method and sodium dodecyl sulfate(SDS)method were used for extracting DNA from the rhizomes of 9 kinds of Aconitum plants before and after preparing. It was investigated that the effects of extracting buffer variety,polyvinyl pyrrolidone(PVP)addition,water-bath time,and DNA sedimentation mode on DNA extraction from the rhizomes of 9 kinds of Aconitum plants before and after preparing,and the effects of two-time polymerase chain reaction(PCR) technology on concentration increasement of ITS2 fragments in Aconitum preparing products.Results The DNA extraction efficiencies were different using interactions of various extracting buffers with waterbath time. It was the best for PCR amplification efficiency on ITS2 in DNA extracted from the crude medicinal materials of 9 kinds of Aconitum plants by 2×CTAB(containing 2.5 mol/L NaCl)with 3-hour water bath,and from the preparing products of 9 kinds of Aconitum plants by 2×CTAB(containing 1.4 mol/L NaCl)with 3-hour water bath. PVP addition or interactions of PVP addition with alterative DNA sedimentation mode has little impact on extracting DNA from crude medicinal materials, but has obvious inhibiting effect on preparing products. The application of two-time PCR promoted the amplification efficiency on ITS2 from preparing products. Conclusion Use of the effective interactions of extracting buffer with water-bath time for DNA extraction and application of twotime PCR technology has effects on promoting preparation efficiency on ITS2 fragments from the rhizomes of Aconitum plants,contributing to providing necessary technical support for DNA barcoding technology application for Aconitum medicinal materials.

关 键 词：乌头属 炮制 DNA提取 二次PCR ITS2 

分 类 号：R285.2[医药卫生—中药学]