鳖甲配方颗粒的位点特异性PCR鉴别研究  

Identification of Trionycis Carapax Formula Granule by Allele-Specific PCR

作　　者：谭斯尹 宋叶 范耀耀 刘闪闪 胡绮萍 罗宇琴 李国卫 TAN Si-yin;SONG Ye;FAN Yao-yao;LIU Shan-shan;HU Qi-ping;LUO Yu-qin;LI Guo-wei(Guangdong Yifang Pharmaceutical Co.,Ltd./Guangdong Key Laboratory of TCM Formula Granule Enterprises,Foshan 528244,China)

机构地区：[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

出　　处：《中药材》2023年第9期2160-2164,共5页Journal of Chinese Medicinal Materials

基　　金：广东省基础与应用基础研究基金自然科学基金项目(2020B1515120033)。

摘　　要：目的:建立一种鳖甲配方颗粒特异性聚合酶链式反应(PCR)鉴别方法。方法:根据中华鳖Trionyx sinensis及其混伪品的细胞色素C氧化酶亚基Ⅰ(COXⅠ)基因序列,筛选出中华鳖的特异性单核苷酸多态性(SNP)位点,设计鳖甲鉴别专属引物BJ-5F及BJ-5R。通过优化PCR反应条件,确定最佳的PCR反应体系,并对该方法进行耐受性、适用性考察和测序验证。结果:当退火温度为60℃,循环33次时,鳖甲药材、标准汤剂冻干粉及配方颗粒均可在约236 bp处得到清晰单一的中华鳖条带,其混伪品及阴性对照品均无此条带。结论:该研究所建立的特异性PCR鉴别方法适用于快速鉴定鳖甲药材、标准汤剂冻干粉及配方颗粒的真伪,且具有专属性强、准确性高、简便易行等特点,为鳖甲配方颗粒质量标准的制定提供了参考依据。Objective:To establish a specific polymerase chain reaction(PCR)identification for Trionycis Carapax formula granule.Methods:Based on the cytochrome C oxidase subunitⅠ(COXⅠ)sequence of Trionyx sinensis and its adulterants,specific primers BJ-5F,BJ-5R were designed according to the specific single nucleotide polymorphisms(SNP)of Trionyx sinensis.The optimal PCR reaction system was determined by optimizing the PCR reaction procedure,and the tolerability,applicability and sequencing of this method were investigated.Results:When the annealing temperature was 60℃and the cycle number was 33,a single discriminant band at about 236 bp of Trionycis Carapax,standard decoction freeze-dried powder and formula granule could be amplified,while no band was found in other adulterants and negative control.Conclusion:The allele-specific PCR method established in this study can quickly and accurately identify the origin of Trionycis Carapax,standard decoction freeze-dried powder and formula granule,it has the characteristics of strong specificity,high accuracy,simplicity and so on,which provides a reference for the formulation of quality standard of Trionycis Carapax formula granule.

关 键 词：鳖甲 配方颗粒 位点特异性PCR 分子鉴定 

分 类 号：R282.74[医药卫生—中药学]