基于DNA熔解曲线分析技术的前胡及其混伪品快速分子鉴定  

Rapid molecular identification of Peucedani Radix and its adulterants by DNA melting curve analysis

作　　者：徐攀[1,2] 吴晓俊 陈如兵 楼柯浪 占靓卉 梁卫青 胡轶娟[1,2] 浦锦宝 XU Pan;WU Xiao-jun;CHEN Ru-bing;LOU Ke-lang;ZHAN Liang-hui;LIANG Wei-qing;HU Yi-juan;PU Jin-bao(Center for Medicinal Resources Research,Zhejiang Academy of Traditional Chinese Medicine,Hangzhou 310007,China;Key Labortory of Research and Development of Chinese Medicine of Zhejiang Province,Hangzhou 310007,China)

机构地区：[1]浙江省中医药研究院药用资源研究中心,杭州310007 [2]浙江省中药新药研发重点实验室,杭州310007

出　　处：《中华中医药杂志》2023年第3期1202-1207,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：中央本级重大增减支项目(No.2060302);国家中医药管理局全国中药资源普查项目(No.GZYKJS-2018-004);浙江省中医药现代化专项项目(No.2020ZX004);浙江省科技计划项目(No.LGF21H280003)。

摘　　要：目的:本研究旨在建立熔解曲线分析方法以实现简便快速直观地鉴别前胡及其混伪品。方法:采用硅胶吸附柱法抽提前胡及其混伪品的总基因组DNA,筛选合适引物,对引物浓度、模板浓度、退火温度和循环次数进行优化,建立前胡及混伪品样品的DNA熔解曲线鉴别方法,并对该方法进行验证。结果:核糖体DNA第二内部转录间隔区(ITS2)为适合的鉴别引物,在引物浓度0.20~0.40μmol/L,DNA模板浓度0.62~15.58 ng/μL,退火温度为53~54℃,循环数为35~45时可获得稳定、均一、特异性的PCR扩增。随后对比熔解曲线熔解温度(Tm值),即可实现前胡药材真伪及掺伪品鉴别,对掺伪品的检测限可低至5%。结论:本方法灵敏度高、特异性好、检测速度快、直观性强,通过进行熔解曲线Tm值比对即可区分不同前胡样品,在该药材的混伪品快速检测方面具有独特的优势。Objective:To establish a melting curve analysis method to identify Peucedani Radix and its adulterants easily,quickly and directly.Methods:The total genomic DNA of Peucedani Radix and its adulterants were extracted by silica gel adsorption column,and the appropriate primers were screened.The primer concentration,template concentration,annealing temperature and cycle times were optimized.The DNA melting curves approach of Peucedani Radix and its adulterants was established and verified.Results:The results showed that ITS2 was a suitable primer,and stable,uniform and specific PCR amplification were performed under the PCR conditions of 0.20-0.40μmol/L primer concentrations,0.62-15.58 ng/μL DNA template,53-54℃annealing temperature and 35-45 cycles.Then the Tm value of the melting curve was compared to realize the identification of the authenticity and adulteration of Peucedani Radix.The detection limit of adulteration could be as low as 5%.Conclusion:The method has the merits of high sensitivity,good specificity,fast detection speed and strong intuition.Different samples can be distinguished by comparing the Tm value of melting curve.It has unique advantages in the rapid detection of mixed counterfeit products of this medicinal material.

关 键 词：熔解曲线 分子鉴定 前胡 混伪品 快速检测 

分 类 号：R282.5[医药卫生—中药学]