Rapid Analysis

Authentication of Agkistrodon acutus based on loop-mediate isothermal amplification*

Expand
  • 1. College of Nursing and Health Management, Lingnan Institute of Technology, Guangzhou 510663, China;
    2. Institute of Analysis, Guangdong Academy of Sciences (China National Analytical Center Guangzhou), Guangzhou 510070, China;
    3. School of Pharmacy, Lingnan Institute of Technology, Guangzhou 510663, China;
    4. Pharmacy Department of Maoming people's Hospital, Maoming 525000, China

Received date: 2023-06-16

  Online published: 2024-06-24

Abstract

Objective: To establish a rapid visual identification method for Agkistrodon acutus based on loop-mediated isothermal amplification (LAMP). Methods: The LAMP assay for the detection of Agkistrodon acutus was developed by designing four primers including two inner primers and two outer primers which according to the specific sequence of Agkistrodon acutus contained in the Chinese Pharmacopoeia. Specificity and sensitivity of the LAMP assay was validated by comparing the polymerase chain reaction (PCR) method included in the Chinese Pharmacopoeia, and using the LAMP assay and the PCR to identify the authenticity of agkistrodon acutus on the market. Results: The LAMP assay established by using those primers showed good performance on specificity and sensitivity. Amplification could be observed in reference crude drug of Agkistrodon acutus and four samples of Agkistrodon acutus from the market,and there was no amplification in one sample of Agkistrodon acutus from the market and eight common adulterants, which was consistent with the identification results of PCR. The sensitivity of the LAMP assay was 0.004 ng·μL-1 and the sensitivity of the PCR assay was 0.4 ng·μL-1, so that the LAMP assay showed higher sensitivity. Conclusion: The result demonstrated that the specific LAMP assay can be used for rapid detection of Agkistrodon acutus.

Cite this article

DENG Hua-ming, XU Xiao-ke, HUANG Qi-hong, LI Zi-hang, LIANG Lin-yuan, YU Zhi-xing, LIANG Yong-heng . Authentication of Agkistrodon acutus based on loop-mediate isothermal amplification*[J]. Chinese Journal of Pharmaceutical Analysis, 2023 , 43(7) : 1263 -1269 . DOI: 10.16155/j.0254-1793.2023.07.21

References

[1] 中华人民共和国药典 2020年版. Vol Ⅰ[S].2020: 388
ChP 2020. Vol Ⅰ[S].2020: 388
[2] 颜克秋, 王剑一, 李格金. 龙胶囊联合经导管肝动脉化疗栓塞术治疗原发性肝癌的疗效和安全性的Meta分析[J].中医肿瘤学杂志, 2021, 3(2):95
YAN KQ, WANG JY, LI GJ. Meta-analysis of randomized controlled trials of Jinlong capsules combined with transcatheter arterial chemoembolization in the treatment of primary liver cancer[J].J Oncol Chin Med, 2021, 3(2):95
[3] 杨通印, 易韦, 文敬. 金龙胶囊联合新辅助化疗治疗浸润性乳腺癌的疗效及多药耐药蛋白表达的变化[J].中华肿瘤杂志, 2019, 41(2):118
YANG TY, YI W, WEN J. Therapeutic effect of Jinlong capsule combined with neoadjuvant chemotherapy on invasive breast cancer and the expression change of multidrug resistance proteins[J].Chin J Oncol, 2019, 41(2):118
[4] LI JW, HAN B, SUN GZ, et al. Chinese patent medicine(Jinlong capsule) for gastric cancer: protocol for a systematic review and meta-analysis[J].Medicine, 2020, 99(23):e20532
[5] 刘滨, 刘维. 蕲蛇治疗类风湿关节炎的临床研究[J].天津中医药, 2016, 33(8):470
LIU B, LIU W. Clinical research of long-noded pit viper on rheumatoid arthritis[J].Tianjin J Tradit Chin Med, 2016, 33(8):470
[6] 汪洋, 马民玉. 风湿祛痛胶囊联合柳氮磺吡啶治疗强直性脊柱炎的临床研究[J].现代药物与临床, 2020, 35(10):2002
WANG Y, MA MY. Clinical study on Fengshi Qutong capsules combined with sulfasalazine in treatment of ankylosing spondylitis[J].Drugs Clin, 2020, 35(10):2002
[7] 罗琳, 伍光辉, 罗寿. 风湿祛痛胶囊联合双氯芬酸钠治疗强直性脊柱炎临床研究[J].中国药业, 2019, 28(14):50
LUO L, WU GH, LUO S, et al. Clinical study on Fengshi Qutong capsules combined with diclofenac sodium in the treatment of ankylosing spondylitis[J].China Pharm, 2019, 28(14):50
[8] NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J].Nucleic Acids Res, 2000, 28(12):63
[9] 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
[10] 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 aspergillum in earthworms using loop-mediated isothermal amplification technology[J].Tianjin J Tradit Chin Med, 2021, 38(4):535
[11] YANG L, WU WR, ZHOU H, et al. Rapid identification of Dendrobium officinale using loop-mediated isothermal amplification (LAMP) method[J].Chin J Nat Med, 2019, 17(5):337
[12] WU L, WANG B, ZHAO MM, 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
[13] 陈卫红, 王竹鑫. 动物类中药的快速经验鉴别[J].中医药导报, 2006, 12(4):80
CHEN WH, WANG ZX. Experience in identification of Chinese crude drug with animal quickly[J].Guiding J Tradit Chin Med Pharmacol, 2006, 12(4):80
[14] 李晓蕾. 金龙胶囊中生物组分的荧光定量PCR鉴定[D].广州: 南方医科大学, 2015
LI XL. Realtime Fluorescent Quantitative PCR Analysis of Biological Ingredient in Jinlong Capsule[D].Guangzhou: Southern Medical University, 2015
[15] 苏畅, 宋茜, 邵鹏柱, 等. 《中国药典》中蕲蛇饮片分子鉴定方法的研究和探讨[J].世界科学技术——中医药现代化, 2016, 18(2):369
SU C, SONG Q, SHAO PZ, et al. Study and discussion on molecular identification of Agkistrodon acutus medicinal slices in Chinese Pharmacopoeia[J].Mod Tradit Chin Med Mater Med World Sci Technol, 2016, 18(2):369
[16] YAU FCF, WONG KL, SHAW PC, et al. Authentication of snakes used in Chinese medicine by sequence characterized amplified region(SCAR)[J] . Biodivers Conserv, 2002, 11(9):1653
[17] 陈康. 基于高分辨熔解曲线技术贵细中药材鉴别的示范应用[D].安徽: 安徽中医药大学, 2016
CHEN K. Demonstration Application of High Resolution Melting Curve Technology on Rare Chinese Herbal Medicines[D].Anhui: Anhui University of Chinese Medicine, 2016
[18] 陈康, 蒋超, 袁媛, 等. 快速PCR方法在蛇类药材真伪鉴别中的应用[J].中国中药杂志, 2014, 39(19):3673
CHEN K, JIANG C, YUAN Y, et al. Application of rapid PCR to authenticate medicinal snakes[J].China J Chin Mater Med, 2014, 39(19):3673
[19] 唐晓晶, 冯成强, 黄璐琦, 等. 蕲蛇及其混淆品高特异性PCR鉴别[J].药物分析杂志, 2006, 26(2):152
TANG XJ, FENG CQ, HUAGN LQ, et al. High specific PCR identification of Agkistrodon acutus Güenther and its adulterants [J].Chin J Pharm Anal, 2006, 26(2):152
[20] LI YN, WANG YS, LI MC, et al. Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene[J].Electron J Biotechnol, 2021, 49: 29
Outlines

/