标准研讨

地鳖的环介导等温扩增快速鉴别*

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  • 1.广东省第二中医院(广东省中医药工程技术研究院) 广东省中医药研究开发重点实验室,广州 510095;
    2.广州中医药大学 第五临床医学院,广州 510405;
    3.广州新华学院 药学院,广州 510520
第一作者 Tel:(020)83482098;E-mail:chenwt009@126.com
**Tel:(020)83482098;E-mail:lisumei124@163.com

收稿日期: 2024-01-18

  网络出版日期: 2024-10-17

基金资助

*广东省中医药局科研项目(20244001);2021年省属科研机构稳定性支持项目(粤财科教[2021]113号)

Rapid authentication of Eupolyphaga sinensis by loop-mediated isothermal amplification*

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  • 1. Guangdong Second Traditional Chinese Medicine Hospital (Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine), Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, Guangdong 510095, China;
    2. The School of the Fifth Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405, China;
    3. School of Pharmacy, Guangzhou Xinhua University, Guangzhou 510520, China

Received date: 2024-01-18

  Online published: 2024-10-17

摘要

目的: 建立基于环介导等温扩增技术(LAMP)的地鳖分子鉴定方法。方法: 针对地鳖COⅠ序列设计特异性引物2对(外引物DB-F3、DB-B3和内引物DB-FIP、DB-BIP),在含有Bst DNA聚合酶和甲基红-苯酚红指示剂的反应体系下于63 ℃下恒温扩增90 min,通过目测检视判断反应结果,与DNA条形码和聚合酶链式反应(PCR)鉴定方法进行对比,考察方法专属性和灵敏度。结果: 11批地鳖样品均由紫红色变为橙黄色,1批冀地鳖、9批金边土鳖和1批伪品龙虱均为紫红色,鉴别结果与DNA条形码,PCR结果一致, LAMP检出限为0.984 ng·mL-1结论: 本项目建立的LAMP检测方法准确,灵敏度高,操作简便快捷,对设备要求低,可应用于地鳖鉴别的快速筛查。

本文引用格式

陈伟韬, 李养学, 江洁怡, 谢灿辉, 贾德政, 刘晓彤, 廖惠娜, 李素梅 . 地鳖的环介导等温扩增快速鉴别*[J]. 药物分析杂志, 2024 , 44(9) : 1529 -1534 . DOI: 10.16155/j.0254-1793.2024-0035

Abstract

Objective: To establish an authentication method for Eupolyphaga sinensis based on loop-mediated isothermal amplification (LAMP). Methods: Two pairs of primers (outer primer DB-F3,DB-B3 and inner primer DB-FIP,DB-BIP) were designed according to Eupolyphaga sinensis COⅠspecific loci. The reaction system containing template, primers, Bst DNA polymerase and methyl red-phenol red indicator amplified at 63 ℃ for 90 min. The LAMP results were determined by visual observation and compared with DNA barcoding and polymerase chain reaction (PCR) methods to investigate the specificity and sensitivity. Results: All 11 batches of the authentic Eupolyphaga sinensis sample tubes colour changed from purple red to orange yellow, while sample tubes of 1 batch of Steleophaga plancyi, 9 batches Opisthoplatia orientalis and 1 batch of the adulterant, Cybister tripunctatus orientalis, remained purple red after the reaction. The LAMP results were consistent with those of DNA barcoding and PCR. The detection limit of LAMP was 0.984 ng·mL-1. Conclusion: The established LAMP method is accurate, sensitive and easy to operate, low equipment required, and can be applied to the rapid screening of the authentication of Eupolyphaga sinensis.

参考文献

[1] 朱良春. 虫类药的应用[M]. 太原:山西科学技术出版社, 1994:45
ZHU LC. The Application of Insect TCM [M]. Taiyuan: Shanxi Science and Technology Publishing House, 1994:45
[2] 王和沪, 王云启. 土鳖虫的本草考证[J]. 中医药导报, 2023, 29(4):52
WANG HH, WANG YQ. Textual research on Tubiechong (Ground Beetle) [J]. Guid J Tradit Chin Med Pharm, 2023, 29(4):52
[3] 常淮阳,史红专,邹洁,等. 雌雄地鳖内在品质及营养成分差异研究[J]. 中国中药杂志, 2022, 47(12):3192
CHANG HY, SHI HZ, ZOU J,et al. Comparison of internal quality and nutrients between male andfemale Eupolyphaga sinensis [J]. China J Chin Mater Med, 2022, 47(12):3192
[4] 国家中医药管理局《中华本草》编委会. 中华本草. 第九册 [M]. 上海:上海科学技术出版社, 1999:151
National Administration of Traditional Chinese Medicine Chinese Materia Medica Editorial Board. Chinese Materia Medica Volume 9 [M]. Shanghai: Shanghai Scientific & Technical Publishers, 1999:151
[5] 广东省食品药品监督管理局. 广东省中药材标准. 第二册 [M]. 广州:广东科学技术出版社, 2011:203
Guangdong Food and Drug Administration. Guangdong Chinese Medicinal Material Standard. Vol 2 [M]. Guangzhou: Guangdong Science & Technology Press, 2011:203
[6] 陈伟韬, 刘梦云, 李养学,等. 金边土鳖薄层色谱鉴别方法的改进[J]. 时珍国医国药, 2020, 31(4):872
CHEN WT, LIU MY, LI YX, et al. Study on improvement of Opisthoplatia orientalis Burm. TLC identification method [J]. J Lishizhen Med Mater Med Res, 2020, 31(4):872
[7] 陈伟韬, 陈昭, 罗文汇,等. 地鳖和金边土鳖的色谱及分子生物学鉴别研究[J]. 今日药学, 2017, 27(8):517
CHEN WT, CHEN Z, LOU WH, et al. Study on identification between Eupolyphaga sinensis Walker and Opisthoplatia orientalis Burm. by chromatography and molecular biology [J]. Pharm Today, 2017, 27(8):517
[8] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12):E63
[9] MORI Y, NOTOMI T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases[J]. J Infect Chemother, 2009, 15(2): 62
[10] 李振庆,吴到检,张大伟,等. 基于毛细管电泳的环介导恒温扩增技术检测方法研究[J]. 分析测试学报, 2016, 35(6):758
LI ZQ, WU DJ, ZHANG DW,et al. Detection of loop-meditated isothermal amplification products by capillary electrophoresis [J]. Instrum Anal, 2016, 35(6):758
[11] 王芳, 董菁, 李艳妮,等. 基于核酸分子光开关的闭管可视化环介导等温扩增检测方法[J]. 分析测试学报, 2022, 41(4):646
WANG F, DONG J, LI YN, et al. A visualized closed-tube detection method for loop-mediated isothermal amplification based on nucleic acid molecular light switch [J]. J Instrum Anal, 2022, 41(4):646
[12] SASAKI Y, NAGUMO S. Rapid identification of Curcuma longa and C. aromatica by LAMP[J]. Biol Pharm Bull, 2007, 30(11): 2229
[13] YANG L, WANG WR, ZHANG H, et al. Rapid identification of Dendrobium officinale using loop-mediated isothermal amplification (LAMP) method[J]. Chin J Nat Med, 2019, 17(5):337
[14] WU L, WANG B, ZHAO M, et al. Rapid identification of Officinal Akebiae Caulis and its toxic adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by loop-mediated isothermal amplification [J]. Front Plant Sci, 2016, 7:887
[15] 王姝涵,隋爱华,韩亚斐,等. 基于环介导等温扩增技术的巴戟天检测方法的建立[J]. 精准医学杂志, 2021, 36(3):217
WANG SH, SUI AH, HAN YF, et al. Establishment of a detection method for Morinda officinalis how based on loop-mediated isothermal amplification [J]. J Precis Med, 2021, 36(3):217
[16] 邓华明,徐晓可,黄启红,等. 基于环介导等温扩增技术的中药材蕲蛇真伪鉴别方法的建立[J]. 药物分析杂志, 2023, 43(7):1263
DENG HM, XU XK, HUANG QH, et al. Authentication of Agkistrodon acutus based on loop-mediated isothermal amplification [J]. Chin J Pharm Anal, 2023, 43(7):1263
[17] 张环宇,谭伟,高晗,等. 中药地龙中参环毛蚓环介导等温扩增快速检测方法[J]. 天津中医药, 2021, 38(4):535
ZHANG HY, TAN W, GAO H, et al. Rapid identification of Pheretima asperglium based on loop-mediated isothermal amplification[J]. Tianjin J Tradit Chin Med, 2021, 38(4):535
[18] ZHAO M, SHI Y, WU L, et al. Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence[J]. Sci Rep, 2016, 6:25370
[19] LI M, WONG YL, JIANG LL, et al. Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd. [J]. Food Chem, 2013, 141 (3):2522
[20] LAI GH, CHAO J, LIN MK, et al. Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification [J]. Int J Mol Sci,2015, 16(1), 1562
[21] CHEN SL, YIN XM, HAN JP, et al. DNA barcoding in herbal medicine: retrospective and prospective[J]. J Pharm Anal,2023, 13(5):431
[22] 许淑莹,王冬梅,欧阳松应. 基于RPA的病原体快速诊断策略[J]. 福建师范大学学报(自然科学版), 2024, 40(1):34
XU SY, WANG DM, OUYANG SY. RPA-based rapid diagnostic strategies for pathogens[J]. J Fujian Norm Univ (Nat Sci Ed), 2024, 40(1):34
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