荧光定量PCR法鉴别蛤蚧定喘丸中醋鳖甲  

Identification of vinegar-quenched Trionycis carapax in Gejie Dingchuan Pills by real-time PCR

作　　者：韩婧 陈香 李叶 沈玉萍[1] 孙小祥 夏国华[1] 杨欢[1] HAN Jing;CHEN Xiang;LI Ye;SHEN Yu-ping;SUN Xiao-xiang;XIA Guo-hua;YANG Huan(College of Pharmacy,Jiangsu University,Zhenjiang 212013,China;Zhenjiang Food and Drug Inspection and Testing Center,Zhenjiang 212003,China;Department of Pharmacy,Affiliated Hospital of Nanjing University of Chinese Medicine,Zhenjiang 212003,China)

机构地区：[1]江苏大学药学院,江苏镇江212013 [2]镇江市食品药品监督检验中心,江苏镇江212003 [3]南京中医药大学附属镇江医院药剂科,江苏镇江212003

出　　处：《中成药》2024年第4期1209-1213,共5页Chinese Traditional Patent Medicine

基　　金：国家自然科学基金项目(82173937);镇江市科技计划项目(SH2023014);南通市基础科学研究和民生科技计划项目(JCZ2022031)。

摘　　要：目的建立蛤蚧定喘丸中醋鳖甲的DNA分子鉴定方法。方法根据鳖、佛罗里达鳖线粒体基因序列的差异设计特异性引物TS和AF,进行荧光定量PCR和方法学考察,然后对市售蛤蚧定喘丸中醋鳖甲进行鉴定。结果含有鳖、佛罗里达鳖的DNA样品仅与相应引物扩增后可检测到荧光信号,有特定温度单一峰,T_(m)分别为77.45、79.72℃。该方法的检测限为10 copies/μL,重复性较高(CV<1%)。10批蛤蚧定喘丸中有9批仅检出鳖DNA,被鉴定为正品;1批同时检出鳖DNA和佛罗里达鳖DNA,被鉴定为掺伪品。结论本研究所建立的方法可快速、可靠地鉴定蛤蚧定喘丸中醋鳖甲炮制品成分,有助于监管含醋鳖甲成方制剂的质量,并为成方制剂中动物药成分的专属性鉴定研究提供新途径。AIM To establish a DNA molecular identification method for vinegar-quenched Trionycis Carapax in Gejie Dingchuan Pills.METHODS Two specific primers(TS and AF)were designed according to the difference of mitochondrial gene sequence between Trionyx sinensis and Apalone ferox,and real-time PCR amplification and method ological investigation were carried out.The established method was used to identify the T.Carapax in commercially available Gejie Dingchuan Pills.RESULTS The fluorescence signals of DNA samples containing T.sinensis and A.ferox could only be detected after amplification with the corresponding primers,and there was a specific temperature peak(T_(m)was 77.45℃or 79.72℃).The assay exhibited a good specificity and high repeatability(CV<1%),and limit of detection was above 10 copies/μL.Among the 10 batches of Gejie Dingchuan Pills,the presence of T.sinensis but not A.ferox were detected in nine of them,which were identified as authentic.Another batch was found to contain both T.sinensis and A.ferox,which was identified as adulterant.CONCLUSION The established method in this study can quickly and reliably identify vinegar-quenched T.Carapax in Gejie Dingchuan Pills,which is helpful to monitor the quality of Chinese Patent Medicines(CPM)containing vinegar-quenched Trionycis Carapax,and provides a new approach for specific identification of animal-derived components in CPM.

关 键 词：蛤蚧定喘丸 醋鳖甲 特异性引物 荧光定量PCR 

分 类 号：R282.5[医药卫生—中药学]