快速分析

基于酶促恒温扩增技术快速鉴别天麻真伪检测方法的建立与应用*

展开
  • 1.北华大学医学技术学院,吉林 132013;
    2.吉林省中药DNA指纹检测技术科技创新中心,吉林 132013;
    3.吉林国安药业有限公司,吉林 132013
第一作者 Tel:18943386083;E-mail:1206225768@qq.com
** Tel:18604498547;E-mail:gaolijun1969@126.com

收稿日期: 2023-07-14

  网络出版日期: 2024-06-20

基金资助

*医药健康产业发展专项(20230401096YY);北华大学研究生创新计划项目【2022】058号;吉林省科技厅重点科技成果转化项目(20170307001YY)

Establishment and application of a rapid authentication test method based on enzymatic recombinase amplification technology for the identification of Gastrodiae Rhizoma*

Expand
  • 1. School of Medical Technology, Beihua Universsity, Jilin 132013, China;
    2. Innovation Center for Detection on DNA Fingerprint of Traditional Chinese Medicine, Jilin 132013, China;
    3. Jilin Guoan Pharmaceutical Limited Company, Jilin 132013, China

Received date: 2023-07-14

  Online published: 2024-06-20

摘要

目的: 基于酶促恒温扩增(enzymatic recombinase amplification, ERA)技术建立一种可快速定性鉴别道地药材天麻真伪的方法。方法: 遵循ERA引物设计原则,应用Oligo 7.0软件根据天麻及其常见伪品的ITS2基因组序列,筛选优化天麻ERA特异性引物,应用Primer Premier 5.0软件设计天麻特异性PCR鉴别引物,通过对ERA、PCR反应体系的优化,最终确定ERA最佳反应时间为17 min,最适反应温度为40 ℃;PCR最优退火温度为57 ℃,循环为32次,对所建立的方法进行灵敏度、特异性验证,并选取中药市场市售天麻样品进行检测。结果: 所设计的天麻ERA鉴别引物与其常见伪品间无交叉反应,特异性良好;重复性结果显示,3次重复性检测结果一致,未出现假阳性与假阴性;该方法对天麻基因组DNA的灵敏度为1 pg·μL-1,比传统PCR灵敏性高;ERA技术能够用于天麻市售样品的快速鉴定,且检测结果与PCR方法相同。结论: 所建立的检测方法操作简单、快速,具有较高的特异性和灵敏度,为道地药材天麻的真伪鉴别提供了一种新手段。

本文引用格式

马秋贺, 马玉贺, 李涛, 刘悦, 柴金军, 徐子强, 刘昂, 高丽君, 夏薇, 李明成, 曲永梅 . 基于酶促恒温扩增技术快速鉴别天麻真伪检测方法的建立与应用*[J]. 药物分析杂志, 2024 , 44(4) : 729 -736 . DOI: 10.16155/j.0254-1793.2024.04.20

Abstract

Objective: To establish a method for the rapid identification of the authenticity of Gastrodiae Rhizoma herbs based on enzymatic recombinase amplification (ERA) technique. Methods: Following the principle of ERA primer design, Oligo 7.0 software was applied to screen and optimize the ERA-specific primers of Tianma based on the ITS2 genome sequences of Gastrodia elata and its common artifacts. Primer Premier 5.0 software was applied to design the specific PCR primers for the identification of Gastrodia elata, and through the optimization of ERA and PCR reaction system, the optimal reaction time for ERA was finally determined to be 17 min, the optimal reaction temperature was 40 ℃, the optimal annealing temperature for PCR was 57 ℃, and the cycle was 32 times, and the established method was verified for sensitivity and specificity, and the samples of asparagus available in the traditional Chinese medicine The sensitivity and specificity of the established method were verified, and the commercially available asparagus samples in the market were selected for testing. Results: The designed primers for the specific identification of ERA in Gastrodia elata did not cross-react with its common forgeries, and the specificity was good, a repeatability results showed that the three repeatability tests were consistent, with no false positives or false negatives; the sensitivity of this method for Gastrodia elata genomic DNA was 1 pg·μL-1, which was higher than that of conventional PCR; the ERA technique can be used for the rapid identification of commercially available samples of Gastrodia elata and the results of the assay are the same as those of the PCR method. Conclusion: The established detection method is simple, rapid, with high specificity and sensitivity, and provides a new means for the authentication of the Gastrodiae Rhizoma.

参考文献

[1] 黄颖, 唐靖雯, 田兴中, 等. 五种不同品系或产地天麻冻干粉抗惊厥及神经保护作用研究[J]. 时珍国医国药, 2021, 32(8):1996
HUANG Y, TANG JW, TIAN XZ, et al. Anticonvulsant and neuroprotective effects of lyophilized powder of five different strains of asparagus of different origins[J]. Lishizhen Med Mater Med Res, 2021, 32(8):1996
[2] 徐帮会, 徐剑, 张永萍, 等. 天麻HPLC指纹图谱及镇痛活性的谱效关系研究[J]. 中药材, 2019, 42(11):2588
XU BH, XU J, ZHANG YP, et al. Correlation analysis between HPLC fingerprint and analgesic activity of Gastrodia elata[J]. J Chin Med Mater, 2019, 42(11):2588
[3] 许鑫, 周欣, 楚世峰, 等. 天麻素治疗缺血性脑卒中导致神经元死亡的作用机制研究[J]. 中国药理学通报, 2020, 36(11):1581
XU X, ZHOU X, CHU SF, et al. Study on the mechanism of gastrodin in the treatment of neuronal death caused by ischemic stroke[J]. Chin Pharmacol Bull, 2020, 36(11):1581
[4] 张大燕, 文欢, 王伟, 等. 天麻对帕金森小鼠神经元保护作用机制的转录组学分析[J]. 中国实验方剂学杂志, 2018, 24(16):73
ZHANG DY, WEN H, WANG W, et al. Transcriptome analysis of protective mechanism of Gastrodiae Rhizoma on neurons of parkinson mice[J]. Chin J Exp Tradit Med Form, 2018, 24(16):73
[5] 虞小静, 张冬月, 李思雨, 等. 天麻水提物HPLC指纹图谱的建立及其真伪鉴别[J]. 中成药, 2018, 40(12):2698
YU XJ, ZHANG DY, LI SY, et al. Establishment of HPLC fingerprints and identification of aqueous extract from Gastrodia elata[J]. Chin Tradit Pat Med, 2018, 40(12):2698
[6] 于碧岩, 于福同, 王贺. 利用稳定同位素技术鉴定天麻真伪[J]. 中华中医药学刊, 2020, 38(9):186
YU BY, YU FT, WANG H. Identification of authenticity of Tianma(Gastrodia elata Bl) by stable isotope technique[J]. Chin Arch Tradit Chin Med, 2020, 38(9):186
[7] 方宣启, 汪霞丽, 王芳斌, 等. 非线性化学指纹图谱鉴别天麻真伪及产地[J]. 食品安全质量检测学报, 2018, 9(17):4693
FANG XQ, WAMG XL, WANG FB, et al. Identification of the authenticity and origin of Gastrodia elata Bl. by non-linear chemical fingerprint[J]. J Food Saf Qual, 2018, 9(17):4693
[8] 李松梅, 孙红洋, 罗静, 等. 基于ITS2条形码的天麻及其伪品DNA分子鉴定[J]. 贵州中医药大学学报, 2023, 45(1):28
LI SH, SUN HY, LUO J, et al. Molecular identification of Gastrodia elata and its counterfeit DNA based on ITS2 barcode[J]. J Guizhou Univ Tradit Chin Med, 2023, 45(1):28
[9] 王慧芳, 喻勇新, 李晨虹, 等. 重组酶聚合酶扩增反应结合侧流层析试纸条技术快速鉴别大西洋鳕鱼制品真伪[J]. 食品与发酵工业, 2022, 48(17):291
WANG HF, YU YX, LI CH, et al. Real-time characterization of Atlantic cod by recombinase polymerase amplification coupled lateral flow dipstick(RPA-LFD)[J]. Food Ferment Ind, 2022, 48(17):291
[10] 魏晓通, 周宇航, 刘扬, 等. 酶促恒温扩增技术快速鉴定食品中牡蛎成分[J]. 食品科技, 2022, 47(11):255
WEI XT, ZHOU YH, LIU Y, et al. Rapid identification of oyster components in food by enzymatic recombinase amplification[J]. Food Sci Technol, 2022, 47(11):255
[11] 沈泓, 袁媛, 陈碧莲, 等. 基于重组酶介导扩增技术的水牛角快速现场鉴别方法[J]. 中国实验方剂学杂志, 2019, 25(17):130
SHEN H, YUAN Y, CHEN BL, et al. On-site identification of medicinal Bubali Cornu by recombinase-mediated amplification technique[J]. Chin J Exp Tradit Med Form, 2019, 25(17):130
[12] 张森豪, 王雪莹, 蔡李萌, 等. 猪流行性腹泻病毒重组酶聚合酶扩增快速诊断方法的建立和应用[J]. 畜牧兽医学报, 2023, 54(6):2498
ZHANG SH, WANG XY, CAI LM, et al. Establishment and application of rapid diagnosis method for RPA of porcine epidemic diarrhea virus[J]. Acta Vet Zootech Sin, 2023, 54(6):2498
[13] 陈德强, 孙碧莲, 李海登, 等. 简单异尖线虫重组酶聚合酶等温扩增检测方法的建立[J]. 中国食品工业, 2023(2):73
CHEN DQ, SUN BL, LI HD, et al. Establishment of an isothermal amplification method for the detection of Anisakis simplex recombinase polymerase[J]. China Food Ind, 2023(2):73
[14] 于继彬, 李俊, 马陈翠, 等. 一种常温核酸扩增反应: 中国, CN109971834A[P]. 2019-07-05
YU JB, LI J, MA CC, et al. A Room Temperature Nucleic Acid Amplification Reaction: China, CN109971834A[P]. 2019-07-05
[15] PIEPENBURG O, WILLAMS CH, ARMES NA, et al. Recombinase Polymerase Amplification: US, US2005112631[P]. 2005-5-26
[16] 史贇学, 王鑫, 王峻, 等. 重组酶恒温扩增技术在动植物源成分分析中的应用与研究进展[J/OL]. 中国食品卫生杂志[2024-02-21]. http://kns.cnki.net/kcms/detail/11.3156.R.20221031.1159.002.html
SHI YX, WANG X, WANG J, et al. Application and research progress of recombinase isothermal amplification technology in the analysis of animal and plant source components[J/OL]. Chin J Food Hyg [2024-02-21]. http://kns.cnki.net/kcms/detail/11.3156.R.20221031.1159.002.html
[17] LI J, MACDONALD J, STETTEN FV. Review: a comprehensive summary of a decade development of the recombinase polymerase amplification[J]. Analyst, 2019, 144(1):31
文章导航

/