药学学报, 2021, 56(9): 2577-2583
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.
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 probe[J]. Acta Pharmaceutica Sinica, 2021, 56(9): 2577-2583.

TaqMan-MGB实时荧光PCR法检测太白贝母及其近缘种的研究
张田1, 陈娇1, 蒋瑞平1, 邹萌1, 仰铁锤2, 付绍兵2, 周嘉裕1*, 廖海1*
1. 西南交通大学生命科学与工程学院, 四川 成都 610031;
2. 青海绿康生物开发有限公司, 青海 西宁 810003
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。
关键词:    TaqMan-MGB探针      太白贝母      ITS1      实时荧光PCR      分子鉴定     
Identification of Fritillaria taibaiensis and its relatives by real-time PCR with a TaqMan-MGB probe
ZHANG Tian1, CHEN Jiao1, JIANG Rui-ping1, ZOU Meng1, YANG Tie-chui2, FU Shao-bing2, ZHOU Jia-yu1*, LIAO Hai1*
1. School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China;
2. Qinghai Lvkang Biological Development Co., Ltd., Xining 810003, China
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.
Key words:    TaqMan-MGB probe    Fritillaria taipaiensis    ITS1    real-time PCR    molecular identification   
收稿日期: 2021-02-14
DOI: 10.16438/j.0513-4870.2021-0230
基金项目: 国家自然科学基金青年科学基金项目(31500276);四川省重点研发项目(2018SZ0061);四川省科技计划项目(2021ZHFP0170);四川省中医药管理局项目(2020JC0128,2021MS116).
通讯作者: 周嘉裕,E-mail:spinezhou@home.swjtu.edu.cn;廖海,E-mail:ddliaohai@home.swjtu.edu.cn
Email: spinezhou@home.swjtu.edu.cn;ddliaohai@home.swjtu.edu.cn
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参考文献:
[1] State Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (中华人民共和国药典)[M]. 2020 Ed. Part I. Beijing:China Medical Science Press, 2020:38-39.
[2] Li Y, Wang YE, Bai YG, et al. A comparative study was conducted on Fritillaria ussuriensis, Fritillaria pallidiflora and Fritillaria cirrhosa based on principal components and antitussive and expectorant effects[J]. Guid J Tradit Chin Med Pharm (中医药导报), 2018, 24:46-56.
[3] Sun T, Peng C. The study of anti-asthmatic effect of Bulbus Fritillariae Cirrhosae cough relieving granule[J]. Lishizhen Med Mater Med Res (时珍国医国药), 2013, 24:1575-1577.
[4] Zhou Y, Ding H, Yan BH, et al. Experimental study on the difference of antitussive and expectorant effects of Fritillaria cirrhosa from different basic sources[J]. Chin J Clin Pharmacol Ther (中国临床药理学与治疗学), 2010, 15:612-616.
[5] Shen L, Ma L, Liu SX, et al. Comparative study on antitussive and expectorant pharmacological effects of Fritillaria unibracteata and Fritillaria taipaiensis[J]. J Pract Tradit Chin Med (实用中医药杂志), 2012, 28:784-785.
[6] Jiang BQ. Identification characteristics of Fritillaria cirrhosa and its similar, confused and counterfeit products[J]. China Pharm (中国药业), 2010, 19:73.
[7] Zhang WJ, Liu W, Wei F, et al. Study on the detection of adulteration of Fritillaria cirrhosa by polymerase chain reactation-restriction fragment length polymorphism[J]. Chin J Pharm Anal (药物分析杂志), 2014, 34:1830-1835.
[8] Hu W, Chen WS, Lin XN, et al. Optimization of polymerase chain reactor-restriction fragment length polymorphism (PCR-RFLP) method for identification of Fritillaria cirrhosa[J]. Chin J Pharm Anal (药物分析杂志), 2017, 37:1716-1720.
[9] Wu D, Guo Q. Detection of other animal species in Placenta Hominis by using Taqman real time PCR[J]. J Chin Med Mater (中药材), 2017, 40:38-41.
[10] Li J, Zhang R, Yu S, et al. Identification of Chinese medicinal materials Stellariae Radix and its adulterants using DNA barcoding[J]. Acta Pharm Sin (药学学报), 2019, 54:937-943.
[11] Lou Q, Xin T, Song J. Application of DNA barcoding technology in the whole industrial chain of traditional Chinese medicine[J]. Acta Pharm Sin (药学学报), 2020, 55:1784-1791.
[12] Zheng H, Deng KY, Chen AQ, et al. Molecular identification and genetic relationship of Fritillaria cirrhosa and related species based on DNA barcode[J]. Acta Pharm Sin (药学学报), 2019, 54:2326-2334.
[13] Zheng M, Chen XQ, Wang S, et al. A TaqMan-MGB real-time RT-PCR assay with an internal amplification control for rapid detection of Muscovy duck reovirus[J]. Mol Cell Probes, 2020, 52:101575.
[14] Watzinger F, Ebner K, Lion T. Detection and monitoring of virus infections by real-time PCR[J]. Mol Aspect Med, 2006, 27:254-298.
[15] Guo LX, Duan LJ, Wang YC, et al. Tanman MGB based real-time PCR method for the detection of Didymella glomerata[J]. Acta Phytopathol Sin (植物病理学报), 2019, 50:97-106.
[16] Liu W, Zhang WJ, Cheng XL, et al. Progress in constituents and isolation and analysis methods of Fritillariae Cirrhosae Bulbus[J]. Asia-Pac Tradit Med (亚太传统医药), 2015, 11:41-46.
[17] Xu CL, Li HJ, Li P, et al. Study on molecular identification method of Fritillaria cirrhosa[J]. J China Pharm Univ (中国药科大学学报), 2010, 41:226-230.
[18] Chen SL, Song JY, Yao H, et al. Strategy and key technique of identification of Chinese herbal medicine using DNA barcoding[J]. Chin J Nat Med (中国天然药物), 2009, 7:322-327.
[19] Xin T, Yao H, Luo K, et al. Stability and accuracy of the identification of Notopterygii Rhizomaet Radix using the ITS/ITS2 barcodes[J]. Acta Pharm Sin (药学学报), 2012, 47:1098-1105.
[20] Yun KY, Xiang L, Wang XY, et al. Identification of Ophiocordyceps sinensis and its adulterants based on portable and CFX96 real-time fluorescent PCR systems[J]. Acta Pharm Sin (药学学报), 2019, 54:746-752.
[21] Guo LX, Duan LJ, Wang CY, et al. TaqMan MGB base real time PCR method for the detection of Didymella glomerata (in Chinese)[J]. Acta Phytopathol Sin (植物病理学报), 2020, 50:97-106.
[22] Zhou GY, Lan H, Li HW. Research on changes of PCR enthalpy in different annealing temperature with DSC[J]. Food Mach (食品与机械), 2011, 27:28-30,118.
[23] You Y, Tataurov AV, Owczarzy R. Measuring thermodynamic details of DNA hybridization using fluorescence[J]. Biopolymers, 2011, 95:472-486.
[24] Xin T, Xu Z, Jia J, et al. Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing[J]. Acta Pharm Sin (药学学报), 2018, 53:488-497.
[25] Zhou CD, Huang ZS. Application of fluorescent quantitative PCR in drug inspection[J]. Tianjin Pharm (天津药学), 2018, 30:65-71.
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