侯飞侠;曹静;王沙沙;王茜;袁媛;彭成;万德光;国锦琳;
HOU Fei-xia;CAO Jing;WANG Sha-sha;WANG Xi;YUAN Yuan;PENG Cheng;WAN De-guang;GUO Jin-lin;Pharmacy College,Chengdu University of Traditional Chinese Medicine,the Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine,Key Laboratory of Systematic Research,Development and Utilization of Chinese Medicine Resources in Sichuan Province—Key Laboratory Breeding Base of Co-founded by Sichuan Province and MOST;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource

• 1:成都中医药大学药学院中药材标准化教育部重点实验室四川省中药资源系统研究与开发利用重点实验室—省部共建国家重点实验室培育基地
• 2:中国中医科学院中药资源中心道地药材国家重点实验室培育基地

摘要(Abstract)：

冬虫夏草为我国传统名贵中药材。资源短缺、价格昂贵和巨大的市场需求导致市售冬虫夏草掺伪掺假现象严重,亟需建立快速有效的鉴别方法。该研究基于实验前期已建立的冬虫夏草快速鉴定SS-PCR方法,按照体外诊断试剂评价标准开发试剂盒,并对试剂盒的特异性、检测限、重复性及保存期4项性能进行了评价。结果显示冬虫夏草质量占混合物质量的1/200(质量分数0.5%以上)以上时可被成功检测。试剂盒对冬虫夏草及其伪品的检测结果显示,仅有冬虫夏草可被成功扩增,并在紫外下显示绿色荧光。该试剂盒37℃温育7 d仍有效,说明4℃保存其在1年内稳定有效。同一批次试剂盒在不同条件下,不同批次试剂盒在相同条件下,其检测结果均一致且准确,表明试剂盒具有良好的批内及批间重复性。将该试剂盒用于市售冬虫夏草及其混伪品的检测,操作简单快速,结果准确可靠,为下一步中药快检试剂盒的商品化奠定了基础。
Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage,expensive price and huge market demand,there are many adulterants of O. sinensis in markets. Therefore,it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis,this study developed a detection kit by optimizing the components and evaluated the specificity,detection limit,repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture,it could be detected successfully. Moreover,only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃for three days,which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions,and in different batch under the same operation conditions gave the same result and accuracy,which showed good repeatability of the kit. It is simple,rapid and accurate to distinguish O. sinensis from its adulterants using the kit,and lays the foundation for commercialization of traditional Chinese medicine fast detection kit.

关键词(KeyWords)： 冬虫夏草;聚合酶链式反应(PCR);检测试剂盒
Ophiocordyceps sinensis;PCR;detection kit

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(30801522,81373920,81541167);; 四川省中医药管理局项目;; 成都市科技局项目

作者(Author): 侯飞侠;曹静;王沙沙;王茜;袁媛;彭成;万德光;国锦琳;
HOU Fei-xia;CAO Jing;WANG Sha-sha;WANG Xi;YUAN Yuan;PENG Cheng;WAN De-guang;GUO Jin-lin;Pharmacy College,Chengdu University of Traditional Chinese Medicine,the Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine,Key Laboratory of Systematic Research,Development and Utilization of Chinese Medicine Resources in Sichuan Province—Key Laboratory Breeding Base of Co-founded by Sichuan Province and MOST;State Key Laboratory of Dao-di Herbs Breeding Base,National Resource

Email:

DOI: 10.19540/j.cnki.cjcmm.20170217.011

参考文献(References)：

• [1]中国药典.一部[S].2015:115.
• [2]Chen Y Q,Wang N,Qu L H,et al.Determination of the anamorph of Cordyceps sinensis inferred from the analysis of the ribosomal DNA internal transcribed spacers and 5.8S r DNA[J].Biochem Syst Ecol,2001(29):597.
• [3]孙超.不同产地冬虫夏草的质量参数测定[J].环境昆虫学报,2015(5):1049.
• [4]徐如英.冬虫夏草的真伪鉴别[J].中国药业,2010,19(24):69.
• [5]杨艳青,段军华.冬虫夏草与其伪品的鉴别[J].世界中西医结合杂志,2012,7(1):31.
• [6]康帅,张继,林瑞超.冬虫夏草的性状和显微鉴定研究[J].药学学报,2013,48(3):428.
• [7]Au Dawn,Wang Lijing,Yang Dajian,et al.Application of microscopy in authentication of valuable Chinese medicine I--Cordyceps sinensis,its counterfeits,and related products[J].Microsc Res Tech,2012,75(1):54.
• [8]徐小东.冬虫夏草的正品与混淆品的鉴别方法分析[J].医药前沿,2015,5(23):309.
• [9]张声俊.红外光谱法对冬虫夏草的三级鉴定和研究[J].山地农业生物学报,2011,30(3):230.
• [10]滕伟卓.蝙蝠蛾拟青霉菌株发酵工艺优化、菌粉质量标准及免疫活性研究[D].长春:吉林大学,2015.
• [11]古今,刘萍,冯建涌.用高效毛细管电泳法鉴别冬虫夏草及其伪品[J].药学服务与研究,2005,5(3):230.
• [12]陈璐.名贵中药冬虫夏草特征性蛋白质成分与鉴定方法研究[D].成都:成都中医药大学,2011.
• [13]李杨.冬虫夏草免疫鉴定技术研究[D].沈阳:辽宁大学,2013.
• [14]中国药典.三部[S].2015:378.
• [15]姜碧清.冬虫夏草及其混淆品、掺杂品、伪品的性状鉴别[J].中国药业,2009,18(8):56.
• [16]刘忠权,丁小余,丁秉中.物种特异性PCR在中药材白花蛇舌草鉴定中的应用[J].南京师大学报:自然科学版,2006,29(3):91.
• [17]唐晓晶,冯成强,黄璐琦,等.蕲蛇及其混淆品高特异性PCR鉴别[J].药物分析杂志,2006(2):152.
• [18]蒋超.中药现场、快速鉴别方法的建立——以金银花为例[D].北京:中国中医科学院,2013.
• [19]唐晓晶,冯成强,黄璐琦,等.高特异性PCR方法鉴别乌梢蛇及其混淆品[J].中国药学杂志,2007,42(5):333.
• [20]Wang H,Kim M K,Kwon W S,et al.Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region[J].J Pharm Biomed Anal,2011,55(5):972.
• [21]龙平,崔占虎,李虔全,等.基于位点特异性PCR的黄芪与红芪鉴别方法研究[J].中国中药杂志,2013,38(16):2581.