中国中药杂志

2017, v.42(05) 896-901

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双阻滞位点特异性PCR鉴别铁皮石斛及其近缘物种
Study on identification of Dendrobium officinale and related species by bidirectional PCR amplification of mismatched and specific alleles

董晓曼;蒋超;袁媛;查良平;彭代银;赵玉洋;
DONG Xiao-man;JIANG Chao;YUAN Yuan;ZHA Liang-ping;PENG Dai-yin;ZHAO Yu-yang;School of Pharmacy,Anhui University of Chinese Medicine;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;Key Laboratory of Modern Traditional Chinese Medicines of Anhui Province,School of Pharmacy,Anhui University of Chinese Medicine;

摘要(Abstract):

建立一种快速、准确鉴别铁皮石斛加工制品的方法。该研究通过对Gen Bank数据库收录的铁皮石斛及其同属近缘物种的ITS序列进行对比分析,根据变异位点设计双阻滞位点特异性鉴别引物,并优化PCR条件,对铁皮石斛及其69种近缘种共362个样品进行扩增和检测。结果显示,在退火温度为60℃的同一PCR反应中,铁皮石斛均扩增出397 bp条带,而其他69种近缘物种均无条带,且该方法灵敏度高,检出限可达0.03 mg·L~(-1)。该文所建立的位点特异性PCR方法可实现铁皮石斛的准确鉴别,具有操作简便、稳定可靠、应用范围广的优点。
Based on r DNA ITS sequences of Dendrobium officinale and the other 69 species of Dendrobium,a pair of dismatched allele-specific diagnostic primers,TPSH-AS1 F and TPSH-AS1 R were designed to authenticate D. officinale from the other species. Thebidirectional PCR amplification were performed using the diagnostic primers with the total DNAs of the original plants or processing products as a template. When the annealing temperature was raised to 60 ℃,only the template DNA of D. officinale could be amplified whereas the diagnostic PCRs of the other Dendrobium species were all negative. Compared with the other authentification methods,the bidirectional PCR amplifications is not only simpler and time-saving but practical and effective.

关键词(KeyWords): 铁皮石斛;位点特异性PCR;分子鉴定
Dendrobium officinale;PCR amplificationof specificalleles;molecularidentification

Abstract:

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基金项目(Foundation): 中医药行业科研专项(201407003)

作者(Author): 董晓曼;蒋超;袁媛;查良平;彭代银;赵玉洋;
DONG Xiao-man;JIANG Chao;YUAN Yuan;ZHA Liang-ping;PENG Dai-yin;ZHAO Yu-yang;School of Pharmacy,Anhui University of Chinese Medicine;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;Key Laboratory of Modern Traditional Chinese Medicines of Anhui Province,School of Pharmacy,Anhui University of Chinese Medicine;

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