基于高分辨率熔解曲线分析技术鉴别丹参及其混伪品
Identification of Salvia miltiorrhiza and Its Adulterants by High-Resolution Melting Analysis
赵景莹 1张玉娟 2柳展基 2赵方洲 2汤慧丽 1王宪昌 2朱彦威 2田北京 2韩金龙 2邬爽 2高春华 2单成钢2
作者信息
- 1. 山东中医药大学药学院,山东济南 250355;山东省农业科学院经济作物研究所,山东济南 250100
- 2. 山东省农业科学院经济作物研究所,山东济南 250100
- 折叠
摘要
本研究旨在建立一种基于高分辨率熔解曲线(high resolution melting,HRM)技术高效鉴别丹参及其混伪品的方法.研究采用硅胶吸附柱法纯化基因组DNA,并针对鼠尾草rDNA ITS2区序列设计特异性引物,通过依次对引物浓度、DNA模板浓度、退火温度和循环次数进行系统优化,建立了ITS2熔解曲线模型,并采用HRM技术对不同来源丹参及其混伪品(甘西鼠尾草、云南丹参和鼠尾草)进行鉴别.结果表明,在最适引物浓度为2.5～10.0 μmol/L,DNA模板浓度为26.95～53.90 ng/μL,退火温度为58℃,循环数为35～45时可获得稳定、均一、特异的PCR扩增曲线.该方法通过对比熔解温度(Tm)即可实现对丹参与甘西鼠尾草、云南丹参及鼠尾草的真伪鉴别,在丹参及其伪品的快速检测方面具有独特的优势.
Abstract
The objective of this study was to establish a new method for identifying Salvia miltiorrhiza and its adulterants effectively based on high-resolution melting(HRM)technology.In this study,the genomic DNA was purified by silica gel column and specific primers were designed based on the rDNA ITS2 sequence of S.officinalis.The melting curve model of ITS2 was established by systematically optimizing the concentra-tions of primer and DNA template,annealing temperature and cycle times,and the HRM analysis was used to identify S.miltiorrhiza and its adulterants(S.przewalskii,S.yunnanensis and S.officinalis).The results showed that the amplification was observed to be stable,uniform and specific under the optimal concentrations of primer and template of 2.5 to 10.0 μmol/L and 26.95 to 53.90 ng/μL,the annealing temperature of 58 ℃,and the cycle number of 35 to 45,respectively.Through comparative analysis of melting temperature(Tm),it was achievable to identify S.miltiorrhiza from S.przewalskii,S.yunnanensis,and S.offiicinalis,showing u-nique advantages in rapid detection of S.miltiorrhiza and its adulterants.
关键词
高分辨率熔解曲线/分子鉴定/丹参/伪品Key words
High-resolution melting curve/Molecular identification/Salvia miltiorrhiza/Adulterants引用本文复制引用
基金项目
山东省农业良种工程项目(2021LZGC008)
山东省现代农业产业技术体系中草药产业体系首席专家项目(SDAIT-20)
济宁市重点研发计划项目(2021ZYNS002)
山东省农业科学院农业科技创新工程项目(GXGC2023D04)
出版年
2024
国家科技期刊平台
NSTL
万方数据