基于DNA熔解曲线分析技术的三七粉分子鉴定

Molecular identification of Radix Notoginseng powder by DNA melt curve analysis

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 develop a melt curve analysis method to identify Radix Notoginseng powder in a fast and easy way targeted on the problem of identifying Radix Notoginseng powder on the market which is mixed with other powders. Methods： Thirty copies of the original plant of P. notoginseng,ten batches of Radix Notoginseng powder,and commonly adulterated powder including rice flour,potato flour,wheat flour,millet flour and soybean powder were collected. The DNA of all the collected samples was extracted; the suitable gene fragments and primers were selected and the melt curve of P. notoginseng was obtained with fakes and adulterants on a 1∶ 1 mix. The test conditions including annealing temperature,cycle index and template concentration were optimized; the method of authenticity identification with DNA melt curve was established and the precision and the replication of the method were investigated. Results： The result showed the melt curve analysis method intuitively identified Radix Notoginseng powder when the premier was psbA- trnH and the test was under the conditions of 58- 62 ℃ annealing temperature,35- 40 cycles,and 0. 78- 483. 20 ng·μL- 1DNA template. The method could identify the authenticity and adulteration of Radix Notoginseng powder by comparing the peak shape of the melt curve without obtaining adulterant information beforehand. Conclusion： Melt curve analysis is fast and intuitive,and suitable to detect adulteration of medicinal materials as it can differentiate DNA of different Radix Notoginseng samples.