摘要
目的基于多重连接探针扩增技术(MLPA)建立一种中药材半夏与其常见伪品虎掌、滴水珠的分子鉴定方法,为半夏药材质量控制提供依据。方法从Genbank数据库下载半夏属核基因组内转录间隔区(ITS)序列,利用MEGA 6.05版软件进行序列比对,依据半夏、虎掌及滴水珠ITS序列中的单核苷酸多态性(SNP)位点设计物种特异性MLPA探针,MLPA反应后采用熔解曲线法对扩增产物进行分析。结果半夏、虎掌和滴水珠样品DNA与物种特异性MLPA探针可分别产生位于84.84,82.76及80.14℃的单一熔解峰,表明探针具有良好特异性;构建的探针体系可对0.1 ng模板DNA进行检测,并可有效检出半夏中掺混1%的虎掌或滴水珠样品。对市场收集样品进行检测,鉴别结果准确,灵敏度高。结论该研究建立的多重连接探针扩增方法可准确鉴别半夏和虎掌、滴水珠,具有特异性强、灵敏度高等特点,可为半夏药材质量控制提供技术支持。
Objective To establish a molecular method for the authentication of Pinellia ternate and its common adulterants Pinellia pedatisecta and Pinellia cordata based on multiplex ligation-dependent probe amplification(MLPA),and to provide a basis for quality control of Pinellia ternate medicinal materials.Methods Internal transcribed spacer(ITS)sequences in the nuclear genome of Pinellia Tenore were downloaded from the Genbank database and then aligned using MEGA 6.05.Species-specific MLPA probes were designed after finding single-nucleotide polymorphism(SNP)sites in the ITS fragments of Pinellia ternate,Pinellia pedatisecta and Pinellia cordata.After the MLPA reaction,the amplification products were analyzed by the melting curve method.Results DNAs and species-specific MLPA probes from Pinellia ternate,Pinellia pedatisecta and Pinellia cordata samples can produce a single melting peak at 84.84,82.76 and 80.14℃,respectively,indicating that the probe has good specificity.The established probes system can not only identify 0.1 ng of template DNA,but also can detect Pinellia ternate samples mixed with 1%Pinellia pedatisecta or Pinellia cordata.The samples collected from the market were identified with accurate results and high sensitivity.Conclusion The MLPA method established in this study can accurately identify Pinellia ternate,Pinellia pedatisecta,and Pinellia cordata,which has the characteristics of strong specificity and high sensitivity,and can provide technical support for the quality control of Pinellia ternate medicinal materials.
作者
莫静
王文斌
程华春
李小芳
刘红
汪波
MO Jing;WANG Wenbin;CHENG Huachun;LI Xiaofang;LIU Hong;WANG Bo(Hubei Institute for Drug Control,Wuhan 430075,China;NMPA Key Laboratory of Quality Control of Chinese Medicine,Wuhan 430075,China;School of Life Science,Hubei University,Wuhan 430062,China;School of Pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China)
出处
《医药导报》
CAS
北大核心
2023年第3期322-328,共7页
Herald of Medicine
基金
湖北省青年拔尖人才培养计划
湖北中药质控标准物质研制及技术平台的建立(2020ACA007)
中国食品药品检定研究院项目(TCM2021-YJ11)。
关键词
半夏
虎掌
滴水珠
多重连接探针扩增技术
中药材鉴定
Pinellia ternate
Pinellia pedatisecta
Pinellia cordata
Multiplex ligation-dependent probe amplification
Identification of traditional Chinese medicine
