Zhao Dan, Zhou Tao, Jiang Wei-Ke, Yuan Yuan, Xiao Cheng-Hong, Zheng Wei
Zhongguo Zhong Yao Za Zhi. 2014 Oct;39(19):3689-94.
To establish a convenient and rapid method for identification of Pseudostellariae Radix by molecular identification, the rDNA-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 x 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 degrees C for 1 min, followed by 30 cycles of denaturation at 95 degrees C for 5 seconds, primer annealing and extension at 56 degrees C for 15 seconds, then it was extension at 72 degrees C 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 x 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.
为通过分子鉴定建立一种简便快速的太子参鉴定方法,对太子参及其伪品的核糖体DNA内转录间隔区(rDNA-ITS)序列进行比对以找出特异性片段。针对该片段设计特异性引物并优化PCR扩增条件。在紫外线下观察用100倍SYBR Green I染色的PCR产物的荧光反应。反应缓冲液的浓度包括5.5 μL DNA Taq聚合酶预混液、10 pmol Tzs-2F和10 pmol Tzs-2R、20 - 80 ng模板DNA,加无菌双蒸水至25 μL。PCR热循环参数如下:95℃预变性1分钟,随后进行30个循环,95℃变性5秒,56℃引物退火和延伸15秒,然后72℃延伸30秒。太子参的荧光反应显示绿色荧光,而伪品则没有。提取、扩增DNA以及分子鉴定的所有步骤可在40分钟内成功完成。该方法能特异性扩增太子参的DNA模板,其产物加1 μL 100倍SYBR Green I在紫外线下可产生绿色荧光。该方法简便准确,可用于中药鉴定。