胡峻;詹志来;袁媛;黄璐琦;刘勇;
HU Jun;ZHAN Zhi-lai;YUAN Yuan;HUANG Lu-qi;LIU Yong;School of Chinese Materia Medica,Beijing University of Chinese Medicine;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

• 1:北京中医药大学中药学院
• 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)：

高分辨率熔解曲线(high resolution melting,HRM)是一种主要用于基因分型和突变扫描的分子诊断技术,在中药真伪鉴定中有着广泛应用前景。该文将高分辨率熔解曲线技术应用于木通药材的鉴别,即选择trn H-psb A通用引物,在退火温度为58℃,35个循环数的条件下,建立正品木通药材的熔解曲线模型,并筛选确定其DNA模板浓度、引物浓度、Mg离子浓度的适宜范围。结果表明,在DNA质量浓度为5~125 mg·L-1、引物浓度0.4μmol·L-1、Mg离子浓度2.0 mmol·L-1的条件下,木通药材的熔解曲线为双峰,其Tm为(81.84±0.16),(85.28±0.16)℃;川木通熔解曲线为单峰,其Tm为(83.22±0.19)℃;关木通熔解曲线为双峰,其Tm为(81.67±0.14),(84.24±0.10)℃。该方法可以实现简单、快速、高通量、可视化的的木通类药材的鉴定。
High resolution melting(HRM),an important technology for genotyping and mutation scanning,has broad prospects in the authentification of traditional Chinese medicine. This paper selected universal trn H-psb A primers and used HRM to establish a new methods for identification of Akebia herbs. PCR was conduct at the annealing temperature of 58 ℃ and 35 cycles. The range of the DNA template concentration,the primer concentration and the Mg2 +ion concentration were further analyzed. The results showed the Tm values of Caulis Akebiae was(81. 84 ± 0. 16),(85. 28 ± 0. 16) ℃ and Caulis Clematidis Armandii was(83. 22 ± 0. 19) ℃ and Caulis Aristolochiae manshuriensis was(81. 67 ± 0. 14) ℃,(84. 24 ± 0. 10) ℃ with 5-125 mg·L- 1DNA template,0. 4 μmol·L- 1primer,2. 0 mmol·L- 1Mg2 +. This method can achieve the authentification of Akebia herbs and is simple,fast,high-throughput,visual.

关键词(KeyWords)： 高分辨率熔解曲线;木通;分子鉴定
high resolution melting;Akebia Herbs;molecular identification

Abstract:

Keywords:

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

作者(Author): 胡峻;詹志来;袁媛;黄璐琦;刘勇;
HU Jun;ZHAN Zhi-lai;YUAN Yuan;HUANG Lu-qi;LIU Yong;School of Chinese Materia Medica,Beijing University of Chinese Medicine;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

Email:

DOI:

参考文献(References)：

• [1]中国药典.一部[S].2010:34.
• [2]国植,徐莉.对广防己、关木通导致的国际大规模中毒事件的反思[J].中草药,2001,32(1):88.
• [3]葛美厅,吴国美,胡双丰.木通、当归、淡竹叶真伪鉴别[J].传统医药,2010,19(4):59.
• [4]谢丽莎,廖月葵,黄茂春.3种木通科木通的生药鉴别研究[J].湖南中医药大学学报,2007(27):142.
• [5]周秦,黄剑林,刘继才.中药木通的鉴别及运用[J].陕西中医,2007,28(11):1456.
• [6]马红梅,张伯礼.不同科属木通比较[J].中国中药杂志,2002,27(6):412.
• [7]和建国,李琛.几种木通类药材的鉴别[J].山西医科大学学报,2004,35(3):256.
• [8]孙萍,罗国安.LC-MS鉴定木通、川木通与关木通[J].中药材,2004,27(12):898.
• [9]陈士林,郭宝林,张贵君,等.中药鉴定学新技术新方法研究进展[J].中国中药杂志,2012,37(8):1043.
• [10]田宗城,李峰,王文龙,等.木通属植物的RAPD研究[J].湖南文理学院学报:自然科学版,2005,17(2):40.
• [11]刘美子,李美妮,姚辉,等.川木通与其混伪品和近缘种的ITS2条形码分子鉴定[J].环球中医药,2011,4(6):446.
• [12]Vossen R H,Aten E,Roos A,et al.High resolution melting analysis(HRMA)—more than just sequence variant screening[J].Hum Mutat,2009,30(6):860.
• [13]Wittwer Carl T.High-resolution DNA melting analysis:advancements and limitations[J].Hum Mutat,2009,30(6):857.
• [14]李茹,廖灿,李东至,等.高分辨熔解曲线分析技术检测中国人常见β地中海贫血基因突变[J].中国优生与遗传杂志,2011(7):19.
• [15]傅新晖,邓艳红,范新娟,等.利用高分辨率熔解曲线分析法检测结直肠癌中KRAS基因突变[J].中华普通外科学文献,2009,3(5):376
• [16]Panagiotis Madesis,Ioannis Ganopoulos,Argiriou Anagnostis,et al.The application of Bar-HRM(barcode DNA-high resolution melting)analysis for authenticity testing and quantitative detection of bean crops(Leguminosae)without prior DNA purification[J].Food Control,2012,25(2):576.
• [17]岩芳,张炜,胡晋,等.高分辨率熔解曲线分析(HRM)及其在植物种质资源鉴定中的应用[J].种子,2013(10):57.
• [18]蒋超,黄璐琦,袁媛,等.酶切-熔解曲线分析:一种新的SNP分型方法及其在中药材鉴定中的应用[J].药学学报,2014,49(4):558.