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川贝母药材分子鉴定方法研究

Molecular method for the identification of Bulbus Fritillariae Cirrhosae

  • 摘要: 川贝母基源鉴定对于其临床用药的安全性、有效性非常重要,但基于性状、显微或化学成分的鉴定方法缺乏专属性,因此,建立专属性强的川贝母分子鉴定方法具有重要意义。本研究在本实验室前期研究工作的基础上,建立了一种适用于川贝母商品药材(干鳞茎)的简便可行的聚合酶链反应-限制性酶切图谱(PCR-RFLP)分子鉴定法,即称取贝母干鳞茎20 mg,依次用75%乙醇和灭菌超纯水清洗后,以新型广谱植物基因组DNA快速提取试剂盒提取基因组DNA,以所提取的DNA为模板进行ITS1区PCR扩增和酶切反应,最后将酶切液进行琼脂糖凝胶电泳,所得图谱显示川贝类核糖体DNA的ITS1区存在限制性内切酶SmaⅠ的酶切位点,在100~200 bp之间有2条清晰的酶切条带,而非川贝类均不能被酶切,没有这两条特征性条带。该方法应用于12批贝母商品药材的鉴定,可将川贝类药材与非川贝类药材区别开来。

     

    Abstract: The origin identification of Bulbus Fritillariae Cirrhosae is essential for clinical safety and efficacy.Previously reported identification methods based on macroscopical,microscopical or chemical characteristics lack specificity,therefore,the molecular identification method is of significance.Based on the continous investigation on the identification of Bulbus Fritillariae Cirrhosae in our laboratory,a simple and feasible PCR-RFLP method was newly developed.The experimental procedure was described as follows: after the pretreatment of 20 mg dried bulbs of Fritillaria with 75% ethanol and sterilized ultrapure water,genomic DNA was extracted with the novel and universal plant genomic DNA extraction kit,then PCR amplification of ITS1 regions and restriction enzyme digestion reaction was carried out successively.The resultant electrophoresis spectrum showed that ITS1 regions of Bulbus Fritillariae Cirrhosae were recognized by restriction enzyme SmaⅠwith providing 2 distinct fragments between 100 bp and 200 bp,while other species could not be digested.Finally,12 batches of commercial Bulbus Fritillariae were correctly differentiated through the above-mentioned method.

     

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