CN109266773A - 一种芪胶升白胶囊原料药材的dna条形码鉴定方法 - Google Patents
一种芪胶升白胶囊原料药材的dna条形码鉴定方法 Download PDFInfo
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Abstract
一种芪胶升白胶囊原料药材的DNA条形码鉴定方法,所述芪胶升白胶囊由大枣、阿胶、血人参、淫羊藿、苦参、黄芪和当归按照下述方法制备而成:以上七味,除阿胶外,当归粉碎成细粉;其余大枣等五味,加水煎煮三次,第一次2小时,第二次1.5小时,第三次1小时,合并煎液,滤过,滤液加入烊化后的阿胶,搅匀,浓缩至75‑85℃下相对密度为1.30的稠膏,加入上述当归细粉,混匀,制成颗粒,干燥,装入胶囊,即得;所述DNA条形码鉴定方法包括对大枣、淫羊藿和当归的鉴定方法。本发明可对芪胶升白胶囊中的原料药材大枣、淫羊藿和当归进行鉴定,能更好地控制该产品的治疗,确保其临床疗效。
Description
技术领域
本发明涉及一种芪胶升白胶囊原料药材的DNA条形码鉴定方法,属于药品技术的领域。
背景技术
芪胶升白胶囊是一种苗医中医互补的民族药,内含棕褐色的颗粒及粉末,味苦,主要由大枣、阿胶、血人参、淫羊藿、苦参、黄芪、当归等组成,具有补血益气的功效,临床上用于治疗气血亏损所引起的头昏眼花、气短乏力、自汗盗汗,以及白细胞减少症等。现有技术中,各药材属内药材物种不易区分,影响了临床用药的有效性和安全性。
发明内容
本发明的目的在于,提供一种芪胶升白胶囊原料药材的DNA条形码鉴定方法。本发明可对芪胶升白胶囊中的原料药材大枣、淫羊藿和当归进行鉴定,能更好地控制该产品的治疗,确保其临床疗效。
本发明采用如下技术方案实现:一种芪胶升白胶囊原料药材的DNA条形码鉴定方法,所述芪胶升白胶囊由大枣240-260份、阿胶240-250份、血人参365-385份、淫羊藿365-385份、苦参240-260份、黄芪740-760份和当归240-260份按照下述方法制备而成:以上七味,除阿胶外,当归粉碎成细粉;其余大枣等五味,加水煎煮三次,第一次2小时,第二次1.5小时,第三次1小时,合并煎液,滤过,滤液加入烊化后的阿胶,搅匀,浓缩至75-85℃下相对密度为1.30的稠膏,加入上述当归细粉,混匀,制成颗粒,干燥,装入胶囊,即得;所述DNA条形码鉴定方法包括对大枣、淫羊藿和当归的鉴定方法。
前所述芪胶升白胶囊原料药材的DNA条形码鉴定方法中,所述大枣的鉴别方法为:取低温干燥大枣果肉38-42 mg,照《中国药典中药材DNA条形码标准序列》中果实类药材DNA提取方法提取得到总DNA,照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点,共15条序列,比对后长度为221 bp,有2个变异位点,分别在88 位点T-C变异,94 位点A-G变异;主导单倍型序列特征如下:
CACAACGTTGCCCCCCATCCCAACCTCGACCTCGAGGCGAAGAGGGGGCGGATGCTGGCCTCCCGTGTGCCACGGTCCGCGGCTGGCCGAAATGCGGGTCCCCGGCGACGAGTGCCGCAGCAATCGGTGGTTGTCCAACCCTCGGCTCCCTGCTGCGTGCGCGGATCGCTGTCGCGGCCCTACAGAGACCCCAATGCGCTGCCAATGCGGCGTCTCCAACG,则为大枣,鉴定结束。
前述芪胶升白胶囊原料药材的DNA条形码鉴定方法中,所述淫羊藿的鉴别方法为:取药材及基原植物样本叶片18-22mg,照《中国药典中药材DNA条形码标准序列》中叶类药材提取方法提取得到总DNA;照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点:如果共10条序列,比对后长度为247 bp,有4个变异位点,分别为35位点的
C-T变异,90位点的T-A变异,229、236位点的A-G变异,主导单倍型序列特征如下:CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为淫羊藿E.brevicornu,鉴定结束;如果共10条序列,比对后长度为247 bp,有5个变异位点,分别为7、43位点的G-A变异,90位点的T-A变异,229位点的A-G变异,245位点的C-T变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为箭叶淫羊藿E. sagittatum,鉴定结束;如果共10条序列,比对后长度为247 bp,有4个变异位点,分别为34、245位点的C-T变异,60位点的T-C变异,229位点的G-A变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTGTAAACGACATTCACTTTG,则为柔毛淫羊藿E. pubescens,鉴定结束;如果序列比对后长度为247 bp,有1个变异位点,为210位点的A-T变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATTATGCCTTTGTTCTCTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCACATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为朝鲜淫羊藿E. koreanum,鉴定结束。
前述芪胶升白胶囊原料药材的DNA条形码鉴定方法中,所述当归的鉴别方法为:取药材38-42mg,照《中国药典中药材DNA条形码标准序列》中根及根茎类药材DNA提取方法提取得到总DNA,照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点:如果共10条序列,比对后长度为229 bp,种内无变异位点,其序列特征如下:CGCATCATCTTTGCCCACAACCACTCACTCCTCGTGGAGCTGTACTGGTATGGGGGCGGAAATTGGCCTCCCGTGCCTTGTTGTGCGGTTGGCGCAAAAGTGAGTCTCCGGCGACGGACGTCGTGACATTGGTGGTTGTAAAATACCCTCATGTCTTGTCGCGCGAATCCGCGTCATCTTAGTGAGCTCAAGGACCCTTAGGCGGCACACACTTTGTGCACTTCGAATG,则为当归A. sinensis,鉴定结束。
发明人进行了大量的实验,以下是本发明方法的部分研究
实验例:
1 大枣鉴定
1.1 材料来源
植物样本共15份,来自新疆、江苏南京市、安徽亳州药市、河南禹州药市、河北安国药市等;原始基原植物采自四川大学华西药学院;对照药材购自中国食品药品检定研究院。
1.2 DNA提取及序列扩增
取低温干燥药材果肉约40 mg,照《中国药典中药材DNA条形码标准序列》中果实类药材DNA提取方法操作。基原植物样本照叶类药材DNA提取方法操作。序列扩增照《中国药典中药材DNA条形码标准序列》中标准流程操作。
1.3 ITS2序列特征
药材共15条序列,比对后长度为221 bp,有2个变异位点,分别在88 位点T-C变异,94位点A-G变异。主导单倍型序列特征如下:
CACAACGTTGCCCCCCATCCCAACCTCGACCTCGAGGCGAAGAGGGGGCGGATGCTGGCCTCCCGTGTGCCACGGTCCGCGGCTGGCCGAAATGCGGGTCCCCGGCGACGAGTGCCGCAGCAATCGGTGGTTGTCCAACCCTCGGCTCCCTGCTGCGTGCGCGGATCGCTGTCGCGGCCCTACAGAGACCCCAATGCGCTGCCAATGCGGCGTCTCCAACG,则为大枣,鉴定结束。
2 淫羊藿鉴定
本品为小檗科植物淫羊藿Epimedium brevicornu Maxim.、箭叶淫羊藿Epimedium sagittatum (Sieb.et Zucc.) Maxim. (《Flora of China》收录名为三枝九叶草Epimedium sagittatum (Sieb. Et Zucc.) Maxim.) 、柔毛淫羊藿Epimedium pubescens Maxim.或朝鲜淫羊藿Epimedium koreanum Nakai的干燥叶。
2.1 材料来源
样本共35份。其中淫羊藿E.brevicornu药材样本来自安徽宣城市,陕西西安市、丹凤县、柞水县和西安植物园;基原植物样本采自中国科学院武汉植物园和西安植物园;对照药材(编号FDC205)购自中国食品药品检定研究院。箭叶淫羊藿E. sagittatum药材样本来自四川成都市、湖北恩施市和安徽宣城市;基原植物样本采自上海辰山植物园。柔毛淫羊藿E. pubescens药材样本来自广州广东市、重庆南川市和陕西丹凤市;基原植物样本(标本号YC0440MT07-09)采自上海辰山植物园。朝鲜淫羊藿E. koreanum药材及基原植物样本均来自吉林通化市和辽宁凤城市。
2.2 DNA提取及序列扩增
取药材及基原植物样本叶片约20 mg,照《中国药典中药材DNA条形码标准序列》中叶类药材DNA提取方法操作。序列扩增照《中国药典中药材DNA条形码标准序列》中标准流程操作。
2.3 ITS2序列特征
(1)淫羊藿E.brevicornu共10条序列,包括药材、基原植物、复核样本,比对后长度为247 bp,有4个变异位点,分别为35位点的C-T变异,90位点的T-A变异,229、236位点的A-G变异。主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG。
(2)箭叶淫羊藿E. sagittatum共10条序列,包括药材、基原植物、复核样本和GenBank序列(GQ434791-2),比对后长度为247 bp,有5个变异位点,分别为7、43位点的G-A变异,90位点的T-A变异,229位点的A-G变异,245位点的C-T变异。主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG 。
(2)柔毛淫羊藿E. pubescens共10条序列,包括药材、基原植物、复核样本和GenBank序列(GQ434793),比对后长度为247 bp,有4个变异位点,分别为34、245位点的C-T变异,60位点的T-C变异,229位点的G-A变异。主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTGTAAACGACATTCACTTTG
(3)朝鲜淫羊藿E. koreanum序列比对后长度为247 bp,有1个变异位点,为210位点的A-T变异。主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATTATGCCTTTGTTCTCTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCACATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG。
3 当归的鉴定
本品为伞形科植物当归Angelica sinensis (Oliv.) Diels. 的干燥根。
3.1 材料来源
样本共10份。药材样本来自重庆市、甘肃省定西市和安徽亳州药市;基原植物样本(标本号PS1205MT01)采自甘肃省定西市;对照药材来自中国食品药品检定研究院。
3.2 DNA提取及序列扩增
取药材约40 mg,照《中国药典中药材DNA条形码标准序列》中根及根茎类药材DNA提取方法操作。基原植物样本照叶类药材DNA提取方法操作。序列扩增照《中国药典中药材DNA条形码标准序列》中标准流程操作。
3.3 ITS2序列特征
当归A. sinensis共10条序列,包括药材、基原植物、复核样本、对照药材和GenBank序列(登录号KF725039),比对后长度为229 bp,种内无变异位点,其序列特征如下:CGCATCATCTTTGCCCACAACCACTCACTCCTCGTGGAGCTGTACTGGTATGGGGGCGGAAATTGGCCTCCCGTGCCTTGTTGTGCGGTTGGCGCAAAAGTGAGTCTCCGGCGACGGACGTCGTGACATTGGTGGTTGTAAAATACCCTCATGTCTTGTCGCGCGAATCCGCGTCATCTTAGTGAGCTCAAGGACCCTTAGGCGGCACACACTTTGTGCACTTCGAATG。
本发明利用基因组中一段公认的、相对较短的DNA序列来进行物种鉴定的一种分子生物学技术。由于不同物种的DNA序列是由腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)四种碱基以不同顺序排列组成,因此,对一定长度的DNA序列进行分析即能够区分不同物种。该技术在传统形态分类学对物种准确鉴定的基础上,通过对样品标准序列进行测序,建立DNA条形码数据库,可实现物种鉴定的标准化。该技术具有以下几大优势:(1)只需选用一个或少数几个基因片段即可对绝大部分物种进行准确鉴定;(2)鉴定过程快速,可以在短时间内鉴定大量样本;(3)重复性和稳定性高;(4)实验过程标准、操作简单,容易实现物种鉴定自动化;(5)可有效缓解分类鉴定人才缺乏的现状;(6)可通过互联网和信息平台对现有物种序列信息进行集中统一管理,并可实现共享。该方法由于不受环境因素的影响以及样品形态和材料部位的限制,可为中药原植物和中药材的品种鉴别提供准确依据,还通过为中药材标本制作一张特殊的基因“身份证”,对物种进行快速、准确的识别与鉴定。
本发明的有益效果在于:本发明方法为芪胶升白胶囊原料药材标本(大枣、淫羊藿和当归)制作一张特殊的基因“身份证”,对物种进行快速、准确的识别和鉴定,可有效控制芪胶升白胶囊的质量,既更有利于生产厂家和监督管理部门对产品质量的监测,也可以为医疗部门和患者的治疗提供更好的保障。因此,本发明可对芪胶升白胶囊中的原料药材大枣、淫羊藿和当归进行鉴定,能更好地控制该产品的治疗,确保其临床疗效。
下面结合实施例对本发明作进一步的说明
具体实施方式
实施例1:
芪胶升白胶囊原料配方:大枣250g、阿胶250g、血人参375g、淫羊藿375g、苦参250g、黄芪750g、当归250g。
芪胶升白胶囊制作工艺:以上七味,除阿胶外,当归粉碎成细粉;其余大枣等五味,加水煎煮三次,第一次2小时,第二次1.5小时,第三次1小时,合并煎液,滤过,滤液加入烊化后的阿胶,搅匀,浓缩至75-85℃下相对密度为1.30的稠膏,加入上述当归细粉,混匀,制成颗粒,干燥,装入胶囊,即得。
鉴定方法:
所述大枣的鉴别方法为:取低温干燥大枣果肉38-42 mg,照《中国药典中药材DNA条形码标准序列》中果实类药材DNA提取方法提取得到总DNA,照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点,共15条序列,比对后长度为221 bp,有2个变异位点,分别在88 位点T-C变异,94 位点A-G变异;主导单倍型序列特征如下:
CACAACGTTGCCCCCCATCCCAACCTCGACCTCGAGGCGAAGAGGGGGCGGATGCTGGCCTCCCGTGTGCCACGGTCCGCGGCTGGCCGAAATGCGGGTCCCCGGCGACGAGTGCCGCAGCAATCGGTGGTTGTCCAACCCTCGGCTCCCTGCTGCGTGCGCGGATCGCTGTCGCGGCCCTACAGAGACCCCAATGCGCTGCCAATGCGGCGTCTCCAACG,则为大枣,鉴定结束。
所述淫羊藿的鉴别方法为:取药材及基原植物样本叶片18-22mg,照《中国药典中药材DNA条形码标准序列》中叶类药材提取方法提取得到总DNA;照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点:如果共10条序列,比对后长度为247 bp,有4个变异位点,分别为35位点的
C-T变异,90位点的T-A变异,229、236位点的A-G变异,主导单倍型序列特征如下:CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为淫羊藿E.brevicornu,鉴定结束;如果共10条序列,比对后长度为247 bp,有5个变异位点,分别为7、43位点的G-A变异,90位点的T-A变异,229位点的A-G变异,245位点的C-T变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为箭叶淫羊藿E. sagittatum,鉴定结束;如果共10条序列,比对后长度为247 bp,有4个变异位点,分别为34、245位点的C-T变异,60位点的T-C变异,229位点的G-A变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTGTAAACGACATTCACTTTG,则为柔毛淫羊藿E. pubescens,鉴定结束;如果序列比对后长度为247 bp,有1个变异位点,为210位点的A-T变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATTATGCCTTTGTTCTCTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCACATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为朝鲜淫羊藿E. koreanum,鉴定结束。
所述当归的鉴别方法为:取药材38-42mg,照《中国药典中药材DNA条形码标准序列》中根及根茎类药材DNA提取方法提取得到总DNA,照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点:如果共10条序列,比对后长度为229 bp,种内无变异位点,其序列特征如下:CGCATCATCTTTGCCCACAACCACTCACTCCTCGTGGAGCTGTACTGGTATGGGGGCGGAAATTGGCCTCCCGTGCCTTGTTGTGCGGTTGGCGCAAAAGTGAGTCTCCGGCGACGGACGTCGTGACATTGGTGGTTGTAAAATACCCTCATGTCTTGTCGCGCGAATCCGCGTCATCTTAGTGAGCTCAAGGACCCTTAGGCGGCACACACTTTGTGCACTTCGAATG,则为当归A. sinensis,鉴定结束。
SEQUENCE LISTING
<110> 贵州汉方药业有限公司
<120> 一种芪胶升白胶囊原料药材的DNA条形码鉴定方法
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 221
<212> DNA
<213> Ziziphus jujuba Mil
<400> 1
cacaacgttg ccccccatcc caacctcgac ctcgaggcga agagggggcg gatgctggcc 60
tcccgtgtgc cacggtccgc ggctggccga aatgcgggtc cccggcgacg agtgccgcag 120
caatcggtgg ttgtccaacc ctcggctccc tgctgcgtgc gcggatcgct gtcgcggccc 180
tacagagacc ccaatgcgct gccaatgcgg cgtctccaac g 221
<210> 2
<211> 247
<212> DNA
<213> 淫羊藿(E.brevicornu)
<400> 2
cgcacagcgt cgctcccacc atgatgcctt tgttctgtta tcgggcaact gcaacgtggc 60
ttgggaagcg gatattggcc ccccgtacct ttgtaggcgc ggccggccta aaattcggcc 120
ctcggcgacg agcgtcacga tcagtggtgg ttgaataacc cctttgtcat agaccggtat 180
cgtgttgttt cgtcgtctat ttgggccata tggacccttg cgtgtcgtat aaacgacatt 240
cactctg 247
<210> 3
<211> 247
<212> DNA
<213> 箭叶淫羊藿(E. sagittatum)
<400> 3
cgcacagcgt cgctcccacc atgatgcctt tgttctgtta tcgggcaact gcaacgtggc 60
ttgggaagcg gatattggcc ccccgtacct ttgtaggcgc ggccggccta aaattcggcc 120
ctcggcgacg agcgtcacga tcagtggtgg ttgaataacc cctttgtcat agaccggtat 180
cgtgttgttt cgtcgtctat ttgggccata tggacccttg cgtgtcgtat aaacgacatt 240
cactctg 247
<210> 4
<211> 246
<212> DNA
<213> 柔毛淫羊藿(E. pubescens)
<400> 4
cgcacagcgt cgctcccacc atgatgcctt tgttctgtta tcgggcaact gcaacgtggc 60
ttgggaagcg atattggccc cccgtacctt tgtaggcgcg gccggcctaa aattcggccc 120
tcggcgacga gcgtcacgat cagtggtggt tgaataaccc ctttgtcata gaccggtatc 180
gtgttgtttc gtcgtctatt tgggccatat ggacccttgc gtgtcgtgta aacgacattc 240
actttg 246
<210> 5
<211> 247
<212> DNA
<213> 朝鲜淫羊藿(E. koreanum)
<400> 5
cgcacagcgt cgctcccacc attatgcctt tgttctctta tcgggcaact gcaacgtggc 60
ttgggaagcg gatattggcc ccccgtacct ttgtaggcgc ggccggccta aaattcggcc 120
ctcggcgacg agcgtcacga tcagtggtgg ttgaataacc cctttgtcat agaccggtat 180
cgtgttgttt cgtcgtctat ttgggccaca tggacccttg cgtgtcgtat aaacgacatt 240
cactctg 247
<210> 6
<211> 229
<212> DNA
<213> 当归(Angelica sinensis (Oliv.) Diels)
<400> 6
cgcatcatct ttgcccacaa ccactcactc ctcgtggagc tgtactggta tgggggcgga 60
aattggcctc ccgtgccttg ttgtgcggtt ggcgcaaaag tgagtctccg gcgacggacg 120
tcgtgacatt ggtggttgta aaataccctc atgtcttgtc gcgcgaatcc gcgtcatctt 180
agtgagctca aggaccctta ggcggcacac actttgtgca cttcgaatg 229
Claims (4)
1.一种芪胶升白胶囊原料药材的DNA条形码鉴定方法,其特征在于:所述芪胶升白胶囊由大枣240-260份、阿胶240-250份、血人参365-385份、淫羊藿365-385份、苦参240-260份、黄芪740-760份和当归240-260份按照下述方法制备而成:以上七味,除阿胶外,当归粉碎成细粉;其余大枣等五味,加水煎煮三次,第一次2小时,第二次1.5小时,第三次1小时,合并煎液,滤过,滤液加入烊化后的阿胶,搅匀,浓缩至75-85℃下相对密度为1.30的稠膏,加入上述当归细粉,混匀,制成颗粒,干燥,装入胶囊,即得;所述DNA条形码鉴定方法包括对大枣、淫羊藿和当归的鉴定方法。
2.如权利要求1所述芪胶升白胶囊原料药材的DNA条形码鉴定方法,其特征在于:所述大枣的鉴别方法为:取低温干燥大枣果肉38-42 mg,照《中国药典中药材DNA条形码标准序列》中果实类药材DNA提取方法提取得到总DNA,照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点,共15条序列,比对后长度为221 bp,有2个变异位点,分别在88 位点T-C变异,94位点A-G变异;主导单倍型序列特征如下:
CACAACGTTGCCCCCCATCCCAACCTCGACCTCGAGGCGAAGAGGGGGCGGATGCTGGCCTCCCGTGTGCCACGGTCCGCGGCTGGCCGAAATGCGGGTCCCCGGCGACGAGTGCCGCAGCAATCGGTGGTTGTCCAACCCTCGGCTCCCTGCTGCGTGCGCGGATCGCTGTCGCGGCCCTACAGAGACCCCAATGCGCTGCCAATGCGGCGTCTCCAACG,则为大枣,鉴定结束。
3.如权利要求1所述芪胶升白胶囊原料药材的DNA条形码鉴定方法,其特征在于:所述淫羊藿的鉴别方法为:取药材及基原植物样本叶片18-22mg,照《中国药典中药材DNA条形码标准序列》中叶类药材提取方法提取得到总DNA;照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点:如果共10条序列,比对后长度为247 bp,有4个变异位点,分别为35位点的
C-T变异,90位点的T-A变异,229、236位点的A-G变异,主导单倍型序列特征如下:CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为淫羊藿E.brevicornu,鉴定结束;如果共10条序列,比对后长度为247 bp,有5个变异位点,分别为7、43位点的G-A变异,90位点的T-A变异,229位点的A-G变异,245位点的C-T变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为箭叶淫羊藿E. sagittatum,鉴定结束;如果共10条序列,比对后长度为247 bp,有4个变异位点,分别为34、245位点的C-T变异,60位点的T-C变异,229位点的G-A变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATGATGCCTTTGTTCTGTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCATATGGACCCTTGCGTGTCGTGTAAACGACATTCACTTTG,则为柔毛淫羊藿E. pubescens,鉴定结束;如果序列比对后长度为247 bp,有1个变异位点,为210位点的A-T变异,主导单倍型序列特征如下:
CGCACAGCGTCGCTCCCACCATTATGCCTTTGTTCTCTTATCGGGCAACTGCAACGTGGCTTGGGAAGCGGATATTGGCCCCCCGTACCTTTGTAGGCGCGGCCGGCCTAAAATTCGGCCCTCGGCGACGAGCGTCACGATCAGTGGTGGTTGAATAACCCCTTTGTCATAGACCGGTATCGTGTTGTTTCGTCGTCTATTTGGGCCACATGGACCCTTGCGTGTCGTATAAACGACATTCACTCTG,则为朝鲜淫羊藿E. koreanum,鉴定结束。
4.如权利要求1所述芪胶升白胶囊原料药材的DNA条形码鉴定方法,其特征在于:所述当归的鉴别方法为:取药材38-42mg,照《中国药典中药材DNA条形码标准序列》中根及根茎类药材DNA提取方法提取得到总DNA,照《中国药典中药材DNA条形码标准序列》中标准流程进行序列扩增,得扩增产物;扩增产物进行测序,根据测序结果统计其单倍型序列变异位点:如果共10条序列,比对后长度为229 bp,种内无变异位点,其序列特征如下:CGCATCATCTTTGCCCACAACCACTCACTCCTCGTGGAGCTGTACTGGTATGGGGGCGGAAATTGGCCTCCCGTGCCTTGTTGTGCGGTTGGCGCAAAAGTGAGTCTCCGGCGACGGACGTCGTGACATTGGTGGTTGTAAAATACCCTCATGTCTTGTCGCGCGAATCCGCGTCATCTTAGTGAGCTCAAGGACCCTTAGGCGGCACACACTTTGTGCACTTCGAATG,则为当归A. sinensis,鉴定结束。
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