蒋超;崔占虎;袁媛;赵玉洋;黄璐琦;
JIANG Chao;CUI Zhan-hu;YUAN Yuan;ZHAO Yu-yang;HUANG Lu-qi;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;Nanyang First People's Hospital;

• 1:中国中医科学院中药资源中心道地药材国家重点实验室培育基地
• 2:南阳市第一人民医院

摘要(Abstract)：

完善快速PCR鉴定检测体系,并建立菟丝子或莱菔子的分子鉴别方法。收集不同地区的菟丝子、莱菔子及其易混淆品材料,所有样品进行总DNA的提取,通过对菟丝子、莱菔子及其易混淆样品ITS片段进行扩增、测序,进行同源比对后根据其变异位点设计特异性鉴别引物,采用2步法进行PCR扩增,从而对菟丝子或莱菔子进行鉴别。通过对影响PCR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仪进行考察,分别获得菟丝子、莱菔子快速PCR反应程序。在稀释后的PCR产物中加入SYBR GreenⅠ染料,正品显示出明亮绿色荧光,而混淆品不显示荧光,且可有效的避免因引物二聚体而存在的假阳性问题。快速PCR方法可以简单快速鉴别菟丝子、莱菔子,为实现药材分子鉴别的现场运用提供技术支撑。
To establish an accurate,rapid and efficient method for authenticating Cuscutae Semen and Raphani Semen by using rapid PCR amplification. The samples of Cuscutae Semen,Raphani Semen and their adulterants were collected. The total DNA of the samples has been extracted,and ITS sequence from Cuscutae Semen,Raphani Semen and their adulterants was amplified by PCR and sequenced directionally. These sequences were aligned by using Clustal W. Specific primers were designed and amplified by two-steps PCR amplification method. The rapid PCR methods for authenticating Cuscutae Semen and Raphani Semen were established by optimizing the denatured and annealing temperature,cycle numbers,and etc. When 100 × SYBR Green I was added in the PCR product,strong green fluorescence was visualized under 365 nm UV lamp whereas adulterants showed no florescence. The results indicated that the rapid PCR method can identify Cuscutae Semen and Raphani Semen rapidly. This study provides the technical support for authentication of Chinese medicinal materials.

关键词(KeyWords)： 快速PCR;菟丝子;莱菔子;分子鉴定;荧光检测
rapid PCR;Cuscutae Semen;Raphani Semen;molecular authentication;fluorescence

Abstract:

Keywords:

基金项目(Foundation): 国家中医药管理局中医药行业科研专项(201407003)

作者(Author): 蒋超;崔占虎;袁媛;赵玉洋;黄璐琦;
JIANG Chao;CUI Zhan-hu;YUAN Yuan;ZHAO Yu-yang;HUANG Lu-qi;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;Nanyang First People's Hospital;

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DOI:

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