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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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DOI: |
KeyWord:LAMP isothermal amplification ?[Ru(bpy)2(dppz)]2+ ?visual detection ?S. aureus |
Author | Institution |
WANG Fang,DONG Jing,LI Yan-ni,XU Qin-feng |
School of Food and Bioengineering,Shaanxi University of Science and Technology,Xi′an ,China |
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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. |
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