A Visualized Closed-tube Detection Method for Loop-mediated Isothermal Amplification Based on Nucleic Acid Molecular Light Switch

WANG Fang; DONG Jing; LI Yan-ni; XU Qin-feng

doi:10.19969/j.fxcsxb.21123104

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A Visualized Closed-tube Detection Method for Loop-mediated Isothermal Amplification Based on Nucleic Acid Molecular Light Switch

Scientific Papers | 更新时间：2022-07-01

- A Visualized Closed-tube Detection Method for Loop-mediated Isothermal Amplification Based on Nucleic Acid Molecular Light Switch
- Journal of Instrumental Analysis Vol. 41, Issue 4, Pages: 646-651(2022)
- 作者机构：
  
  陕西科技大学食品与生物工程学院，陕西西安 710021
- 作者简介：
- 基金信息：
- DOI：10.19969/j.fxcsxb.21123104
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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(04):646-651.

WANG Fang,DONG Jing,LI Yan-ni,et al.A Visualized Closed-tube Detection Method for Loop-mediated Isothermal Amplification Based on Nucleic Acid Molecular Light Switch[J].Journal of Instrumental Analysis,2022,41(04):646-651.
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(04):646-651. DOI： 10.19969/j.fxcsxb.21123104.

WANG Fang,DONG Jing,LI Yan-ni,et al.A Visualized Closed-tube Detection Method for Loop-mediated Isothermal Amplification Based on Nucleic Acid Molecular Light Switch[J].Journal of Instrumental Analysis,2022,41(04):646-651. DOI： 10.19969/j.fxcsxb.21123104.

摘要

该研究采用核酸分子“光开关”［Ru（bpy）,2,（dppz）］,2+,作为环介导等温扩增（LAMP）荧光染料，建立了一种快速、直观的闭管可视化LAMP检测方法。为避免高浓度染料对LAMP反应造成的强烈抑制和开盖导致的气溶胶交叉污染，该文采用蜡封反应管进行检测，通过直接观察扩增产物DNA与［Ru（bpy）,2,（dppz）］,2+,结合后产生的强烈红色荧光信号判定结果。该方法对金黄色葡萄球菌DNA的检出限可低至20拷贝/反应。方法特异性强，整个可视化检测过程可在1 h内完成，有望广泛应用于环境检测、食品安全、临床诊断等领域的现场快速核酸检测。

Abstract

Compared with polymerase chain reaction（PCR） nucleic acid amplification，loop-mediated isothermal amplification（LAMP） does not require thermal cycling instruments，which is more suitable for rapid on-site detection．However，the small Stoke′s shift of currently used organic fluorescent dye for visualized LAMP leads to insufficient color differentiation．In this study，the nucleic acid molecule“light switch”［Ru（bpy）,2,（dppz）］,2+, was exploited as a LAMP fluorescent dye to establish a rapid visualized closed-tube method for LAMP detection．Visualization detection requires a high concentration of dye，but its adding before amplification reaction will strongly inhibit LAMP reaction，while its adding after reaction by opening the lid will lead to a serious aerosol cross contamination．Therefore，wax was chosen to seal the［Ru（bpy）,2,（dppz）］,2+, at the bottom of the tube，and the LAMP amplification products were visually detected by using the strong red fluorescence emission of ［Ru（bpy）,2,（dppz）］,2+, in the presence of double-strand DNA．The ,S.aureus, DNA could be detected as low as 20 copies/reaction with strong specificity，and the whole visual detection process could be completed within 1 h．It could be widely used for the rapid on-site nucleic acid detection in the fields of environmental detection，food safety and clinical diagnosis．

关键词

环介导等温扩增法（LAMP）［Ru（bpy）2（dppz）］2+可视化检测金黄色葡萄球菌

Keywords

LAMP isothermal amplification［Ru（bpy）2（dppz）］2+visual detectionS. aureus

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