摘要
目的建立一种龟甲配方颗粒高分辨熔解曲线(HRM)技术鉴别方法。方法根据乌龟Chinemys reevesii及其混伪品的细胞色素C氧化酶亚基Ⅰ(cytochrome oxidase subunitⅠ,COI)基因序列上的单核苷酸多态性(single nucleotide polymorphisms,SNP)位点设计鉴别引物,扩增产物直接进行HRM分析,并优化反应体系,根据最佳条件进行适用性验证。结果在模板DNA质量浓度为0.62~374.88 ng/μL、引物浓度为0.1~0.4μmol/L、退火温度为62℃、循环数为45个的条件下,龟甲饮片和配方颗粒的熔解曲线为单峰,其Tm均值分别为(82.39±0.09)、(82.38±0.05)℃,而混伪品和醋龟甲配方颗粒未有扩增。结论该研究建立的高分辨熔解曲线鉴别方法可快速鉴别龟甲原料和配方颗粒的真伪,以及区分其生品和炮制品配方颗粒,为龟甲配方颗粒的质量标准研究提供了理论依据。
Objective To establish a high-resolution melting curve(HRM)identification for Testudinis Carapax et Plastrum(TCP)formula granule.Methods Based on the cytochrome C oxidase subunitⅠ(COI)sequence of Chinemys reevesii and its adulterant,specific primers were designed according to the specific single nucleotide polymorphisms(SNP)of Chinemys reevesii.The products were analyzed by HRM directly,the reaction was optimized and verified based on the optimal conditions.Results When the DNA template concentration was 0.62~374.88 ng/μL,the primer concentration was 0.1-0.4μmol/L,the annealing temperature was 62℃and the cycle number was 45,the Tm value of TCP herbal decoction pieces and formula granule were(82.39±0.09)and(82.38±0.05)℃,respectively.No band was found in other adulterants and vinegar TCP formula granule.Conclusion The HRM method can quickly identify TCP crude medicine,herbal decoction pieces and formula granule,and also distinguish formula granule of TCP and its processed products,which provides a theoretical basis for the quality standard of Testudinis Carapax et Plastrum formula granule.
作者
谭斯尹
宋叶
刘闪闪
范耀耀
罗宇琴
李国卫
TAN Siyin;SONG Ye;LIU Shanshan;FAN Yaoyao;LUO Yuqin;LI Guowe(Guangdong Yifang Pharmaceutical Co.,Ltd./Guangdong Key Laboratory of TCM Formula Granule Enterprises,Foshan 528244,China)
出处
《广东药科大学学报》
CAS
2024年第3期70-75,共6页
Journal of Guangdong Pharmaceutical University
基金
国家工信部2022年产业技术基础公共服务平台项目-中药全产业链质量技术服务平台(2022-230-221)
2022年佛山市南海区重点领域科技攻关专项(南科﹝2023﹞20号-18)。
关键词
龟甲
配方颗粒
高分辨熔解曲线(HRM)
分子鉴定
Testudinis Carapax et Plastrum
Formula Granule
high-resolution melting curve
molecular identification
