中国中药杂志

2015, v.40(12) 2304-2308

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基于熔解曲线分析技术的木通类药材的分子鉴别
HRM identification of Chinese medicinal materials Mutong

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;

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

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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;

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