| 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 |
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| 基金项目:国家自然科学基金资助项目（22074085）；国家重点研发计划子课题（2019YFC1606302） |
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| 中文摘要: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。 |
| 中文关键词: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 |
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| A Visualized Closed-tube Detection Method for Loop-mediated Isothermal Amplification Based on Nucleic Acid Molecular Light Switch |
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| 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． |
| Key Words:LAMP isothermal amplification ?［Ru（bpy）2（dppz）］2+ ?visual detection ?S. aureus |
| 引用本文：王芳，董菁，李艳妮，徐秦峰.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):646-651. |
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