人参属中药材高分辨率熔解曲线鉴定及其方法学考察  

Identification of high resolution melting curves of Panax species and methodological investigation

作　　者：陈康[1,2] 蒋超[1] 金艳[1] 赵玉洋 黄璐琦[1] 袁媛[1] 

机构地区：[1]道地药材国家重点实验室培育基地,中国中医科学院中药资源中心,北京100700 [2]安徽中医药大学,合肥230038

出　　处：《药物分析杂志》2017年第1期64-73,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：中医药行业科研专项“常用大宗中药材质量现场快速检测技术研究”(201407003)

摘　　要：目的:建立基于高分辨率熔解曲线鉴别人参属中药材的方法,并进行系统性方法学考察。方法:采集不同产地的人参属中药材人参、西洋参、三七以及市场上常见混伪品竹节参、珠子参、羽叶三七。所有样品提取总DNA,筛选合适的引物,构建人参属正品中药材熔解曲线。同时对不同比例混合样品进行了鉴别,对该方法的灵敏性与特异性、重复性、稳健性、检出限进行了系统性考察。结果:选择psbA-F/trnH-R引物,在模板质量浓度1.6~200 ng·μL^(-1),退火温度为54~60℃,引物浓度为0.1~0.3)μmol·L^(-1)范围内,对人参属中药材共75份样品进行高分辨熔解曲线分析人参、西洋参、三七、羽叶三七、竹节参、珠子参等均获得正确稳定的分析结果。结论:高分辨熔解曲线依据分型分析可以区分人参属中药材,并对其混伪品进行检测。在此基础上本文提出了中药分子鉴定方法学研究标准程序,为进一步规范中药分子鉴定方法学研究提供依据。Objective： To establish a method for the identification of Panax species based on high resolution melting （ HRM ） , and to carry out systematic methodological study. Methods ： Panax species such as ： Panax ginseng, Panax quinquefolium, Panax notoginseng, Panax japonicus, Panax japonicas vat. major, Panax psettdoginseng vat. bipinnatifidus were collected. The DNA of all the collected samples were extracted; the suitable primers were selected and the melting curve for Panax species was developed. The fakes and adulterants of different mix were also tested. Molecular identification, including the sensitivity, specificity, repeatability, robustness, and the limit of detection was systematically investigated. Results： A total of 75 samples of Panax ginseng were selected by using primer psbA-F/trnH-R. The template concentration was 1.6-200 ng·μL-1, primer concentration for the psbA-F/trnH-R primer was 0.1-0.3μmol· L-1, and annealing temperature was 54-60℃. The analysis of high-resolution melting curve on Panax species can obtain the correct stable identification. Conclusion： High resolution melting curve analysis can distinguish Panax species by genotyping, and its adulterants can be detected. On this basis, this paper presents a standard method for the molecular identification of traditional Chinese medicine, which provides a basis for further standardizing the molecular identification of traditional Chinese medicine.

关 键 词：中药材 DNA分析 人参属 掺伪品 分子鉴定 高分辨率熔解曲线 方法学考察 

分 类 号：R917[医药卫生—药物分析学]