地鳖的环介导等温扩增快速鉴别  

Rapid authentication of Eupolyphaga sinensis by loop-mediated isothermal amplification

作　　者：陈伟韬 李养学 江洁怡 谢灿辉 贾德政 刘晓彤 廖惠娜 李素梅 CHEN Wei-tao;LI Yang-xue;JIANG Jie-yi;XIE Can-hui;JIA De-zheng;LIU Xiao-tong;LIAO Hui-na;LI Su-mei(Guangdong Second Traditional Chinese Medicine Hospital(Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine),Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine,Guangzhou,Guangdong 510095,China;The School of the Fifth Clinical Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;School of Pharmacy,Guangzhou Xinhua University,Guangzhou 510520,China)

机构地区：[1]广东省第二中医院(广东省中医药工程技术研究院)广东省中医药研究开发重点实验室,广州510095 [2]广州中医药大学第五临床医学院,广州510405 [3]广州新华学院药学院,广州510520

出　　处：《药物分析杂志》2024年第9期1529-1534,共6页Chinese Journal of Pharmaceutical Analysis

基　　金：广东省中医药局科研项目(20244001);2021年省属科研机构稳定性支持项目(粤财科教[2021]113号)。

摘　　要：目的:建立基于环介导等温扩增技术(LAMP)的地鳖分子鉴定方法。方法:针对地鳖COⅠ序列设计特异性引物2对(外引物DB-F3、DB-B3和内引物DB-FIP、DB-BIP),在含有Bst DNA聚合酶和甲基红-苯酚红指示剂的反应体系下于63℃下恒温扩增90 min,通过目测检视判断反应结果,与DNA条形码和聚合酶链式反应(PCR)鉴定方法进行对比,考察方法专属性和灵敏度。结果:11批地鳖样品均由紫红色变为橙黄色,1批冀地鳖、9批金边土鳖和1批伪品龙虱均为紫红色,鉴别结果与DNA条形码,PCR结果一致,LAMP检出限为0.984 ng·mL^(-1)。结论:本项目建立的LAMP检测方法准确,灵敏度高,操作简便快捷,对设备要求低,可应用于地鳖鉴别的快速筛查。Objective:To establish an authentication method for Eupolyphaga sinensis based on loop-mediated isothermal amplification(LAMP).Methods:Two pairs of primers(outer primer DB-F3,DB-B3 and inner primer DB-FIP,DB-BIP)were designed according to Eupolyphaga sinensis COⅠspecific loci.The reaction system containing template,primers,Bst DNA polymerase and methyl red-phenol red indicator amplified at 63℃for 90 min.The LAMP results were determined by visual observation and compared with DNA barcoding and polymerase chain reaction(PCR)methods to investigate the specificity and sensitivity.Results:All 11 batches of the authentic Eupolyphaga sinensis sample tubes colour changed from purple red to orange yellow,while sample tubes of 1 batch of Steleophaga plancyi,9 batches Opisthoplatia orientalis and 1 batch of the adulterant,Cybister tripunctatusorientalis,remained purple red after the reaction.The LAMP results were consistent with those of DNA barcoding and PCR.The detection limit of LAMP was 0.984 ng·mL^(-1).Conclusion:The established LAMP method is accurate,sensitive and easy to operate,low equipment required,and can be applied to the rapid screening of the authentication of Eupolyphaga sinensis.

关 键 词：地鳖 环介导等温扩增技术 DNA条形码 聚合酶链式反应 中药材鉴别 目视检查 快速鉴别 

分 类 号：R917[医药卫生—药物分析学]