中国中药杂志

2009, v.34(23) 3013-3016

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位点特异性PCR方法的建立及对近源种鹿茸药材的鉴别研究
Establishment of allele-specific diagnostic PCR method for identification of antlers

王学勇;刘春生;张蓉;黄璐琦;崔光红;
WANG Xueyong1,LIU Chunsheng1,ZHANG Rong1,HUANG Luqi2,CUI Guanghong2 (1.College of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100102,China;2.Institute of Chinese Meteria Medica,China Academy of Chinese Sciences,Beijing 100700 China)

摘要(Abstract):

目的:针对正品鹿茸与近源种鹿茸药材饮片鉴别的难点,建立一种简便、快速、准确的近源种鹿茸药材分子鉴别方法。方法:根据鹿科鹿属种动物Cytb全序列比对分析,设计1对能准确鉴别正品鹿茸(包括梅花鹿和马鹿)和其他近源种鹿茸的引物,在此基础上建立鹿茸药材位点特异性PCR鉴别方法,并对影响PCR结果的主要因素退火温度、Taq用量、循环次数以及模板用量和重复性等进行方法学考察和优化。结果:在方法学考察的基础上,在25μL PCR反应体系,退火温度为65℃的条件下,能准确扩增出约323 bp大小的阳性DNA条带,经测序验证结果表明,系正品鹿茸原动物的Cytb基因序列片段,而伪品鹿茸未扩增出条带。结论:所建立的位点特异性PCR方法,能将正品鹿茸与其他近源种鹿茸药材鉴别开来,具有较高的特异性、重复性,可广泛应用于鹿类药材的鉴别。
Objective:To establish a convenient,quick and accurate molecular method for the identification of crude drugs of antlers due to the difficult discrimination between the genuine antler and its adulterants.Method: According to the alignment analysis of full length sequences of Cytb gene from closely relate species of Cervus,one pair of allele-specific diagnostic PCR primers was designed.Factors such as annealing temperature,dosage of polymerase,times of cycles and dosage of template DNA that influence the PCR results were also investigated.Result: Based on the study mentioned above,about 323 bp positive band was amplified under the annealing temperature of 65 ℃ in the total volume of 25 μL PCR reaction using the genuine antler DNA as the template.Sequencing results proved that the positive band was the fragment of Cytb gene from both C.elaphus Linnaeus and C.nippon Temminck.Conclusion: The established method,with higher specificity and reproducibility,could accurately differentiate genuine antler from its adulterants and would be widely used in Cervus related Chinese crude drugs′ identification.

关键词(KeyWords): 位点特异性PCR;近源种鹿茸药材;鉴别
Allele-specific diagnostic PCR;antlers from closely relate species of Cervus;identification

Abstract:

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基金项目(Foundation): 《中国药典》2010版一部标准研究项目(YD-195-1)

作者(Author): 王学勇;刘春生;张蓉;黄璐琦;崔光红;
WANG Xueyong1,LIU Chunsheng1,ZHANG Rong1,HUANG Luqi2,CUI Guanghong2 (1.College of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100102,China;2.Institute of Chinese Meteria Medica,China Academy of Chinese Sciences,Beijing 100700 China)

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