广东药科大学学报2024，Vol.40Issue(3) ：70-75.DOI:10.16809/j.cnki.2096-3653.2024030401

龟甲配方颗粒的高分辨熔解曲线技术鉴别研究

Study on the identification of Testudinis Carapax et Plastrum Formula Granule by high-resolution melt-ing curve analysis

谭斯尹 宋叶 刘闪闪 范耀耀 罗宇琴 李国卫
广东药科大学学报2024，Vol.40Issue(3) ：70-75.DOI:10.16809/j.cnki.2096-3653.2024030401
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  • 维普
  • 万方数据

龟甲配方颗粒的高分辨熔解曲线技术鉴别研究

Study on the identification of Testudinis Carapax et Plastrum Formula Granule by high-resolution melt-ing curve analysis

谭斯尹 1宋叶 1刘闪闪 1范耀耀 1罗宇琴 1李国卫1
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作者信息

  • 1. 广东一方制药有限公司/广东省中药配方颗粒企业重点实验室,广东 佛山 528244
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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0.1～0.4 μmol/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.

Abstract

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 0.1-0.4 μmol/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.

关键词

龟甲/配方颗粒/高分辨熔解曲线(HRM)/分子鉴定

Key words

Testudinis Carapax et Plastrum/Formula Granule/high-resolution melting curve/molecular identification

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基金项目

国家工信部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项目(2022)(2022-230-221)

佛山市南海区重点领域科技攻关专项(2022)(南科[2023]20号-18)

出版年

2024
广东药科大学学报
广东药学院

广东药科大学学报

CSTPCD
影响因子：0.698
ISSN：1006-8783
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