中国中药杂志

2015, v.40(04) 619-623

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基于熔解曲线分析技术的鹿茸药材分子鉴别
Molecular identification of hairy antler by analysis of high resolution melting

陈康;蒋超;袁媛;黄璐琦;金艳;
CHEN Kang;JIANG Chao;YUAN Yuan;HUANG Lu-qi;JIN Yan;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;Anhui University of Chinese Medicine;

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。文章将高分辨率熔解曲线技术应用于鹿茸真伪鉴别,利用COⅠ序列,在退火温度为60℃,45个循环数的条件下,建立正品鹿茸药材熔解曲线模型,并筛选确定其DNA模板浓度、引物浓度、Mg2+浓度的适宜范围。结果表明,鹿茸药材在模板DNA质量浓度为10~100 mg·L-1、引物浓度0.2μmol·L-1,Mg2+浓度2.0 mmol·L-1的条件下,梅花鹿熔解曲线为双峰,其Tm分别为(81.96±0.07),(84.51±0.03)℃;马鹿熔解曲线为双峰,其Tm为(82.58±0.13),(85.95±0.05)℃。该方法可以实现简单、快速、高通量、可视化的鹿茸药材真伪鉴定。
High resolution melting( HRM),an important technology for genotyping and mutation scanning,has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal COⅠ primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 ℃ and 45 cycles. The range of the DNA template concentration,the primer concentration and the Mg2 +ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were( 81. 96 ± 0. 07),( 84. 51 ± 0. 03) ℃ and Cervus elaphus was( 82. 58 ± 0. 13),( 85. 95 ± 0. 05) ℃ with10-100 mg·L- 1DNA template,0. 2 μmol·L- 1primer,2. 0 mmol·L- 1Mg2 +. This method can authenticate of hairy antler and is simple,fast,high-throughput,visualization.

关键词(KeyWords): 高分辨熔解曲线;鹿茸药材;分子鉴定
high resolution melting;hairy antler;molecular identification

Abstract:

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基金项目(Foundation): 国家杰出青年科学基金项目(81325023);; 中医药行业专项(2014007003)

作者(Author): 陈康;蒋超;袁媛;黄璐琦;金艳;
CHEN Kang;JIANG Chao;YUAN Yuan;HUANG Lu-qi;JIN Yan;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;Anhui University of Chinese Medicine;

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