张田, 陈娇, 蒋瑞平, 邹萌, 仰铁锤, 付绍兵, 周嘉裕, 廖海. TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究J. 药学学报, 2021,56(9): 2577-2583. doi: 10.16438/j.0513-4870.2021-0230
引用本文: 张田, 陈娇, 蒋瑞平, 邹萌, 仰铁锤, 付绍兵, 周嘉裕, 廖海. TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究J. 药学学报, 2021,56(9): 2577-2583. doi: 10.16438/j.0513-4870.2021-0230
ZHANG Tian, CHEN Jiao, JIANG Rui-ping, ZOU Meng, YANG Tie-chui, FU Shao-bing, ZHOU Jia-yu, LIAO Hai. Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probeJ. Acta Pharmaceutica Sinica, 2021,56(9): 2577-2583. doi: 10.16438/j.0513-4870.2021-0230
Citation: ZHANG Tian, CHEN Jiao, JIANG Rui-ping, ZOU Meng, YANG Tie-chui, FU Shao-bing, ZHOU Jia-yu, LIAO Hai. Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probeJ. Acta Pharmaceutica Sinica, 2021,56(9): 2577-2583. doi: 10.16438/j.0513-4870.2021-0230

TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究

Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe

  • 摘要: 采用TaqMan-MGB探针实时荧光PCR方法对太白贝母及其近缘种进行分子鉴定研究。提取太白贝母及其近缘种的DNA,依据ITS1区序列,利用MEGA7.0软件进行比较分析,找出太白贝母及其近缘种的变异位点,通过Primer Premier 6.0软件设计一对特异性引物和TaqMan-MGB探针。在Roche LightCycler 96系统中,该方法对太白贝母DNA模板的检测下限为0.002 39 ng·μL-1,最适Tm值区间为60与61℃。特异性鉴定研究表明,该方法对太白贝母有良好的特异性,能与暗紫贝母等其余13种不同贝母基原植物明显区分。TaqMan-MGB实时荧光PCR方法可实现对太白贝母及其近缘种的准确鉴定,为太白贝母资源的合理开发、中药材市场的管理和中药生产企业的原料监管提供技术支撑。

     

    Abstract: The molecular identification of Fritillaria taibaiensis and its relatives was studied by real-time PCR with a TaqMan-MGB probe. DNA was extracted from F. taibaiensis and its relatives. According to the sequence of ITS1 region, the mutation sites of F. taipaiensis and its related species were identified by MEGA7.0 software. The specific primers (a pair) and a TaqMan-MGB probe were designed by Primer Premier 6.0 software. In the Roche LightCycler 96 system, the lowest limit of detection for F. taipaiensis DNA template was 0.002 39 ng·μL-1, and the optimal Tm value range was 60 and 61℃. Specificity identification showed that the method had good specificity for F. taipaiensis, as it could be distinguished from other 13 different Fritillaria species including F. unibracteata. Since this method could accurately identify F. taipaiensis and its related species, it provides technical support for rational development of F. taipaiensis resources, management of Chinese medicinal market and supervision of raw materials in Chinese medicine manufacturing enterprises.

     

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