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  

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

作　　者：王芳 董菁 李艳妮 徐秦峰 WANG Fang;DONG Jing;LI Yan-ni;XU Qin-feng(School of Food and Bioengineering,Shaanxi University of Science and Technology,Xi′an 710021,China)

机构地区：[1]陕西科技大学食品与生物工程学院,陕西西安710021

出　　处：《分析测试学报》2022年第4期646-651,共6页Journal of Instrumental Analysis

基　　金：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)。

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。Compared with polymerase chain reaction(PCR)nucleic acid amplification,loopmediated 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 

分 类 号：O657.3[理学—分析化学] O629.7[理学—化学]