中国中药杂志

2014, v.39(19) 3673-3677

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快速PCR方法在蛇类药材真伪鉴别中的应用
Application of rapid PCR to authenticate medicinal snakes

陈康;蒋超;袁媛;黄璐琦;李曼;
CHEN Kang;JIANG Chao;YUAN Yuan;HUANG Lu-qi;LI Man;Anhui University of Chinese Medicine;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

摘要(Abstract):

为优化获得一种准确、快速、高效鉴别药典所载蛇类药材(乌梢蛇,金钱白花蛇,蕲蛇)真伪品的方法。该研究以蛇类药材经典PCR鉴定方法为基础,采用碱裂解法提取基因组总DNA,加入特异性PCR引物,采用两步法进行PCR扩增,从而对蛇类药材及其混淆品进行鉴别。通过对影响PCR反应时间的退火温度、变性温度、退火时间、变性时间、循环次数等因素进行优化,并对不同型号PCR仪和Taq酶进行考察,分别获得乌梢蛇、金钱白花蛇、蕲蛇快速PCR反应程序。在PCR产物中加入SYBR Green I染料,正品显示出明亮绿色荧光,而混淆品不显示荧光。所建立的蛇类药材快速PCR真伪检测方法可在30~45 min完成,为蛇类药材现场快速鉴定提供技术支持。
To obtained an accurate,rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia( Zaocysd humnades,Bungarus multicinctus,Agkistrodon acutus),a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized.When 100 × SYBR Green I was added in the PCR product,strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

关键词(KeyWords): 快速PCR;蛇类中药材;分子鉴定;荧光检测
rapid PCR;medical snakes;molecular authentication;fluorescence

Abstract:

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基金项目(Foundation): 中医药行业科研专项(201407003)

作者(Author): 陈康;蒋超;袁媛;黄璐琦;李曼;
CHEN Kang;JIANG Chao;YUAN Yuan;HUANG Lu-qi;LI Man;Anhui University of Chinese Medicine;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

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