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筛选获得草珊瑚与金粟兰鉴别的特异位点并建立双位点特异性PCR方法,用于鉴别草珊瑚与金粟兰叶片以及两者的混杂品。采集不同产地的草珊瑚18份、金粟兰6份,所有样品提取总DNA,并对草珊瑚与金粟兰干燥叶片进行DNA提取。通过对其ITS片段进行扩增、测序,并搜索GenBank数据库中收录的草珊瑚与金粟兰的ITS序列,进行同源比对后根据其变异位点设计特异性鉴别引物,构建多重PCR体系,并优化PCR条件,对草珊瑚与金粟兰叶片进行快速分子鉴定。构建了多重PCR鉴别体系,只经一个PCR反应,能扩增出草珊瑚580 bp的特异性鉴别条带及金粟兰470 bp的特异性片段,可快速鉴别草珊瑚与金粟兰叶片,并可判断相互是否混杂。使用双位点特异性PCR技术可以有效快速鉴别草珊瑚与金粟兰叶片,并可判断相互是否混杂。
Screening for specific sites identified in the coral and the Coturnum and double-site-specific PCR method was established for the identification of grass coral and Opuntia microphylla leaves and a mixture of the two. Eighteen samples of Sarcandra glabrata from different habitats were collected, and six copies of Cotinus coggygria were collected. Total DNA was extracted from all the samples and DNA extracted from the dried leaves of Sarcandra glabra and Coturnum annuus. ITS ITS fragment was amplified and sequenced, and the ITS sequences of S. przewalskii and P. tangutorum collected in GenBank database were searched. After homologous comparison, specific primers were designed according to their variation sites to construct multiplex PCR system and optimize PCR conditions, the rapid identification of the grass coral and Chamomile leaves. A multiplex PCR identification system was constructed. Only one PCR reaction can amplify a 580 bp specific discriminating band and a 470 bp specific fragment of C. cinnabarinus, which can quickly identify the leaves of the grass coral and the Coturnum annuus and judge whether they are mutual Mixed. The use of two-site-specific PCR technology can effectively and quickly identify the grass coral and Opuntia tabacum leaves, and can determine whether each other mixed.