木通与川木通及关木通的快速PCR鉴别

Authentication of Mutong,Chuan-Mutong and Guan-Mutong by Rapid PCR

目的:建立一种准确、快速、高效鉴别木通、川木通及关木通的分子鉴别方法。方法:采集不同产地的木通、川木通及关木通基原植物,所有样品进行总DNA的提取,通过对木通、川木通及关木通样品trn L-trn F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,从而对木通、川木通及关木通进行鉴别。结果: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仪进行考察,分别获得木通、川木通及关木通快速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。结论:快速PCR方法可以简单快速鉴别木通、川木通及关木通,为实现药材分子鉴别的现场运用提供技术支撑。

Objective： To establish an accurate,rapid and efficient method for authenticating Mutong,ChuanMutong and Guan-Mutong by using PCR amplification of specific alleles. Methods： The samples of Mutong,Chuan-Mutong and Guan-Mutong were collected. The total DNA of the samples has been extracted,and trn L-trn F sequence from Mutong,Chuan-Mutong and Guan-Mutong 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. Results： The rapid PCR methods for authenticating Mutong, Chuan-Mutong and Guan-Mutong 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 without. Conclusion： The results indicated that the rapid PCR method can identify Mutong,Chuan-Mutong and Guan-Mutong rapidly. This study provides the technical support for authentication of Chinese medicinal materials.