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目的:建立冬虫夏草与人工发酵菌丝体的鉴别方法。方法:通过聚合酶链式反应(PCR),扩增基因组上的核糖体基因内转录间隔区(ITS),继而用限制性内切酶XhoⅠ对该聚合酶链式反应产物进行酶切,将酶切产物进行琼脂糖凝胶电泳及紫外成像;通过聚合酶链式反应扩增线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)的编码基因,将PCR产物进行琼脂糖凝胶电泳及紫外成像。结果:冬虫夏草ITS的PCR产物能够被XhoⅠ酶切成2个片段。而5种人工发酵菌丝体中,有4种不能够被酶切;由于来源于冬虫夏草无性型中华被毛孢,百令胶囊可被酶切且切割条带大小与冬虫夏草组一致。冬虫夏草样品全部扩增得到COⅠ基因(COⅠ)片段,而5种人工发酵菌丝体均未扩增出该片段。结论:结合聚合酶链式反应限制性内切酶片段长度多态性法以及COⅠ聚合酶链式反应扩增,可以鉴别冬虫夏草与人工发酵菌丝体。
Objective: To establish the identification method of Cordyceps sinensis and artificial fermentation mycelium. Methods: The ribosomal gene internal transcribed spacer (ITS) was amplified by polymerase chain reaction (PCR), then the restriction enzyme XhoⅠ was used to digest the polymerase chain reaction product, and the enzyme The products were subjected to agarose gel electrophoresis and UV imaging. The gene encoding mitochondrial cytochrome C oxidase subunit Ⅰ (COⅠ) was amplified by polymerase chain reaction (PCR) and subjected to agarose gel electrophoresis and UV imaging. Results: The PCR products of Cordyceps sinensis ITS could be digested by Xho Ⅰ into two fragments. Among the five kinds of artificial fermented mycelia, four could not be digested; due to C. sinensis derived from Cordyceps sinensis, Bailing capsule could be digested and the size of the cutting band was consistent with Cordyceps sinensis group. Cordyceps sinensis samples were all amplified CO Ⅰ gene (CO Ⅰ) fragment, and five kinds of artificial fermentation mycelium did not amplify the fragment. Conclusion: Cordyceps sinensis and artificial fermentation mycelium can be identified by polymerase chain reaction-restriction fragment length polymorphism and COⅠ polymerase chain reaction amplification.