中国中药杂志

2014, v.39(19) 3689-3694

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太子参药材的快速分子鉴定
Rapid molecular identification of Pseudostellariae Radix

赵丹;周涛;江维克;袁媛;肖承鸿;郑伟;
ZHAO Dan;ZHOU Tao;JIANG Wei-ke;YUAN Yuan;XIAO Cheng-hong;ZHENG Wei;Guiyang College of Traditional Chinese Medicine;State Key Laboratory of Dao-di Herbs,Chinese Materia Medica Resource Center,China Academy of Chinese Medicinal Sciences;

摘要(Abstract):

为建立一种简便快捷的太子参分子鉴定方法,对太子参药材及其混伪品的r DNA-ITS片段进行比对分析,找出特异性片段,设计太子参鉴别引物,并优选扩增条件,扩增产物经100×SYBR Green I染色后紫外光下观察。结果显示,PCR扩增反应液(包括DNA Taq聚合酶预混液5.5μL,Tzs-2F,Tzs-2R各10 pmol,DNA模板2080 ng,双重灭菌蒸馏水加至25μL)经95℃预变性1 min,95℃变性5 s,56℃退火延伸15 s,30个循环,72℃后延伸30 s后,在扩增产物中加入1μL100×SYBR Green I混匀后于紫外光下呈绿色荧光的为太子参,混伪品无绿色荧光出现。40 min内即可完成DNA提取、扩增等整个鉴定过程。该方法能特异性扩增太子参DNA,紫外光下肉眼观察即可鉴定药材的真伪,且操作简捷、结果准确、利于推广,可为快速鉴定中药材的真伪提供参考。
To establish a convenient and rapid method for identification of Pseudostellariae Radix by molecular identification,the r DNA-ITS sequences of Pseudostella riaheterophylla and its adulterants had been aligned to find out specific fragment. The specific primers against the fragment were designed and the PCR amplification conditions were optimized. The fluorescence reaction of the PCR products colored by 100 × SYBR Green I was observed under UV. The concentration of reaction buffer included 5. 5 μL DNA Taq polymerase premix,10 pmol Tzs-2F and 10 pmol Tzs-2R,20-80 ng template DNA,and plus double sterile distilled water to 25 μL. The PCR thermal profile was as follows: predenaturation at 95 ℃ for 1 min,followed by 30 cycles of denaturation at 95 ℃ for 5 seconds,primer annealing and extension at 56 ℃ for 15 seconds,then it was extension at 72 ℃ for 30 seconds. The fluorescence reaction of Pseudostellariae Radix showed green fluorescence,while adulterants had not. Extraction,amplification DNA and all steps of molecular identification could be completed successfully in 40 minutes. The approach could amplify DNA template of Pseudostellariae Radix specificity,and its product with 1 μL 100 × SYBR Green I could engender green fluorescence under UV. The method was simple and accurate,so it could be used for identification of Chinese traditional medicine.

关键词(KeyWords): 太子参;分子鉴定;特异性PCR;SYBR Green I
Pseudostellariae Radix;molecular identification;specific PCR;SYBR Green I

Abstract:

Keywords:

基金项目(Foundation): 中医药行业科研专项(201407003);; 贵州省中药现代化科技产业研究开发专项(黔科合ZY字[2013]3001号);; 贵州省教育厅普通高等学校特色重点实验室建设(黔教合KY字[2013]108号)

作者(Authors): 赵丹;周涛;江维克;袁媛;肖承鸿;郑伟;
ZHAO Dan;ZHOU Tao;JIANG Wei-ke;YUAN Yuan;XIAO Cheng-hong;ZHENG Wei;Guiyang College of Traditional Chinese Medicine;State Key Laboratory of Dao-di Herbs,Chinese Materia Medica Resource Center,China Academy of Chinese Medicinal Sciences;

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