全蝎分子鉴定及其配方颗粒的位点特异性PCR鉴别  

Site-specific PCR identification of animal species and formula particles of Scorpio

作　　者：段宝丽 陈天韵 施丹丹 尤春雪 蒋超[2] DUAN Bao-li;CHEN Tian-yun;SHI Dan-dan;YOU Chun-xue;JIANG Chao(Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392,China;State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]天津农学院动物科学与动物医学学院,天津市农业动物繁育与健康养殖重点实验室,天津300392 [2]中国中医科学院中药资源中心,道地药材品质保障与资源持续利用全国重点实验室,北京100700

出　　处：《中国中药杂志》2024年第4期942-950,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(83073972);中国中医科学院中药资源中心自主选题研究项目(ZZXT202212)。

摘　　要：全蝎是我国临床常用动物药材,《中国药典》规定全蝎的唯一基原为钳蝎科动物东亚钳蝎,但目前我国蝎目物种丰富,全蝎药材及其深加工制品都存在混伪现象,其中全蝎深加工制品在加工过程中,其显微特征可能不完整或丢失,使得基原鉴别变得困难。该研究基于东亚钳蝎与其他蝎物种的形态和氧化酶亚基1(COⅠ)序列,构建ML系统发育树进行物种鉴定,从而发现全蝎主要混伪品为细尖狼蝎;使用特异性聚合酶链式反应(PCR)鉴定方法,通过COⅠ序列的差异SNP位点设计东亚钳蝎特异性鉴别引物,并筛选出全蝎药材与其配方颗粒的稳定引物区间,对全蝎药材及配方颗粒进行鉴定。通过优化PCR反应体系,当退火温度61℃、循环次数30次时,东亚钳蝎及其配方颗粒均可扩增出约150 bp明亮的特异性条带,其他混伪品均无条带。并对该方法的适应性进行考察,全蝎药材到冻干粉再到配方颗粒以及市售配方颗粒均可扩增出约150 bp的条带。研究结果表明,该方法可用于全蝎药材及其配方颗粒真伪鉴别,有利于完善全蝎配方颗粒的质量控制体系,为其临床应用提供保障。Scorpio,a commonly used animal medicine in China,is derived from Buthus martensii as recorded in the Chinese Pharmacopoeia.China harbors rich species of Scorpionida and adulterants exist in the raw medicinal material and deep-processed products of Scorpio.The microscopic characteristics of the deep-processed products may be incomplete or lost during processing,which makes the identification difficult.In this study,the maximum likelihood(ML) tree was constructed based on the morphology and cytochrome C oxidase subunit I(COⅠ) to identify the species of Scorpio products.The results showed that the main adulterant of Scorpio was Lychas mucronatus.According to the specific SNP sites in the COⅠ sequence of B.martensii,the stable primers were designed for the identification of the medicinal material and formula granules of Scorpio.The polymerase chain reaction(PCR) at the annealing temperature of 61 ℃ and 30 cycles produced bright specific bands at about 150 bp for both B.martensii and its formula particles and no band for adulterants.The adaptability of the method was investigated,which showed that the bands at about 150 bp were produced for Scorpio medicinal material,lyophilized powder,and formula granules,and commercially available formula granules.The results showed that the established method could be used to identify the adulterants of Scorpio and its formula granules,which could help to improve the quality control system and ensure the safe clinical application of Scorpio formula granules.

关 键 词：全蝎 配方颗粒 聚合酶链式反应 系统发育树 物种鉴别 

分 类 号：R282.5[医药卫生—中药学]