基于熔解曲线峰形特异性鉴别川贝母真伪  

Identification of Fritillaria cirrhosa Bulbus Based on DNA Melting Curve Peak Shape of Real-Time PCR

作　　者：陈慕媛[1] 李辉标[1] 唐洪梅[1] 闫雪[1] 陈洁[1] 林霖[2] 陈国培[2] 朱成杰 CHEN Muyuan;LI Huibiao;TANG Hongmei;YAN Xue;CHEN Jie;LIN Lin;CHEN Guopei;ZHU Chengjie(The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405 Guangdong,China;Shenzhen Academy of Metrology & Quality Inspection,Shenzhen 518131 Guangdong,China)

机构地区：[1]广州中医药大学第一附属医院,广东广州510405 [2]深圳市计量质量检测研究院,广东深圳518131

出　　处：《中药新药与临床药理》2019年第5期592-597,共6页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：广东省中医药局科研项目(20172045);广东省医院药学研究基金(2016A08)

摘　　要：目的探讨基于熔解曲线峰形及退火温度(Tm值)在川贝母真伪鉴别中的应用方法。采用《中国药典》2015年版聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)鉴别法检测川贝母样品。使用CLUSTALX软件比对NCBI-Genbank数据库下载的川贝母及其常见伪品特征序列,设计并验证引物与实时荧光PCR,检测市售样品。结果川贝母熔解曲线呈尖锐峰且Tm值为80.98℃,与常见伪品峰形有明显区别。结论此研究利用熔解曲线峰形特异性鉴定川贝母,实验操作简单,鉴定结果直观。Objective To explore the application of Real-Time PCR (RT-PCR) DNA melting curve peak shape and its annealing temperature(Tm value) in identifying Fritillaria cirrhosa bulbus.Methods DNA samples were detected using PCR-RFLP method referring to Chinese Pharmacopoeia.DNA sequences of Fritillaria cirrhosa bulbus and its common pseudo-products from the NCBI-Genbank database were collected and compared by CLUSTAL software.Suitable primers were designed and verified.The melting-curve PCR method verified by reference medicine were used to detect DNA of commercial samples.Results Fritillaria cirrhosa bulbus can be identified by a single sharp peak at the annealing temperature of 80.98 ℃,that is significantly different from those of pseudo-products.Conclusion The study suggests that the method of RT-PCR with DNA melting curve peak shape analysis is convenient and intuitive for detecting Fritillaria cirrhosa bulbus.

关 键 词：川贝母 鉴别 熔解曲线 实时荧光PCR 

分 类 号：R284.1[医药卫生—中药学]