中国中药杂志

2012, v.37(24) 3752-3757

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基于双向位点特异性PCR的金银花真伪鉴别方法研究
Authentication of Lonicera japonica using bidirectional PCR amplification of specific alleles

蒋超;张雅华;陈敏;袁媛;林淑芳;吴志刚;
JIANG Chao,ZHANG Ya-hua,CHEN Min,YUAN Yuan*,LIN Shu-fang,WU Zhi-gang(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

摘要(Abstract):

目的:筛选获得金银花真伪鉴别SNP位点并建立双向位点特异性PCR方法,用于鉴别金银花和其常见伪品以及两者的混杂品。方法:通过对GenBank收录的忍冬属植物叶绿体trnL-trnF序列进行对比分析,获得金银花真伪鉴别SNP位点;并依据该SNP位点设计特异性引物,对84份金银花基原植物及其市售饮片、伪品进行双向位点特异性PCR扩增,并根据真伪品的特异性条带进行金银花药材鉴别。结果:退火温度为61℃时,正品均出现468 bp的条带,红腺忍冬、华南忍冬、灰毡毛忍冬、黄褐毛忍冬、水忍冬、金银忍冬、郁香忍冬、新疆忍冬、繁果忍冬等9个伪品均出现324 bp的条带,在正品DNA中掺入5%以上伪品时同时出现正品和伪品条带。结论:双向位点特异性PCR可以鉴别金银花真伪品及两者的混杂样品。
Objective: To identify SNP in flos Lonicerae,and authenticate Lonicera japonica from its adulterants and the mixture by using bidirectional PCR amplification of specific alleles(Bi-PASA).Method: SNP of L.japonica and its adulterants was identified by using ClustulW to align trnL-trnF sequences of the Lonicera genus from GenBank database.Bi-PASA primer was designed and the PCR reaction systems including annealing temperature optimized.Optimized result was performed in 84 samples to authenticate L.japonica,its adulterants and the mixture.Result: When the annealing temperature was 61 ℃,DNA from L.japonica would be amplified 468 bp whereas PCR products from all of the 9 adulterants were 324 bp.The established method also can detect 5% of intentional adulteration DNA into L.japonica.Conclusion: The Bi-SPASA could authenticate L.japonica from its adulterants and the mixture.

关键词(KeyWords): 双向位点特异性PCR;金银花;分子鉴定
bidirectional PCR amplification of specific alleles;Lonicera japonica;molecular authentication

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基金项目(Foundation): 国家自然科学基金项目(81001605);; 中药制药过程新技术国家重点实验室开放基金项目(SKL2010M0101)

作者(Author): 蒋超;张雅华;陈敏;袁媛;林淑芳;吴志刚;
JIANG Chao,ZHANG Ya-hua,CHEN Min,YUAN Yuan*,LIN Shu-fang,WU Zhi-gang(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

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