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目的:针对市售三七粉掺入其他粉末导致鉴别困难的问题,建立熔解曲线分析方法,以实现简便、快速鉴别三七粉的目的。方法:采集不同产地的三七原植物30株,市售成药三七粉10批以及大米、土豆、小麦、小米、大豆粉末等常见掺假粉末。所有样品提取总DNA,筛选适合片段及引物,分别构建三七、伪品、三七与伪品等比例混合样品DNA的熔解曲线。对退火温度、循环次数以及模板浓度进行优化,建立三七粉DNA熔解曲线分析真伪鉴别方法,并对其精密度和重复性进行考察。结果:选择psbA-trnH作为鉴别引物,在退火温度为58~62℃、循环数为35~40条件下,DNA模板0.78~483.20 ng·μL-1范围内进行熔解曲线分析,可通过熔解曲线峰形比对,在未获得伪品信息的情况下进行三七粉真伪及掺杂品鉴别。结论:熔解曲线分析技术依据熔解曲线的峰形和Tm值大小即可区分不同三七样品的DNA,其直观的特点在该药材掺假品快速检测方面具有独特的优势。
OBJECTIVE: To solve the problem of difficult identification by incorporating other powders into the market of Panax notoginseng, the melting curve analysis method was established to achieve the purpose of identifying Panax notoginseng powder conveniently and rapidly. Methods: Thirty plants of Panax notoginseng from different areas were collected. Ten batches of Panax notoginseng powder and 10 common batches of adulterated powder such as rice, potato, wheat, millet and soybean were collected. The total DNA was extracted from all the samples, and the suitable fragments and primers were screened to construct the melting curves of the DNAs of mixed samples such as notoginseng, counterfeit, notoginseng and counterfeit. The annealing temperature, the number of cycles and the concentration of template were optimized to establish the method of authenticity identification of DNA melting curve of Panax notoginseng powder, and its precision and repeatability were investigated. Results: Using psbA-trnH as the differential primer, the melting curve was analyzed in the range of 0.78 ~ 483.20 ng · μL-1 with the annealing temperature of 58 ~ 62 ℃ and the number of cycles of 35 ~ 40, Shape comparison, in the case of counterfeit information is not carried out under the authenticity and notoginseng powder doping identification. Conclusion: The melting curve analysis technique can distinguish the DNA of different Panax notoginseng samples according to the peak shape and the Tm value of the melting curve. Its intuitive features have unique advantages in rapid detection of the adulterated product.