Quantification of Colorimetric Loop-mediated Isothermal Amplification Process

A We propose a novel quantitative assay for colorimetric loop-mediated isothermal amplification (LAMP) based on the shift of UV-vis spectral absorbance of Eriochrome black T (EBT) dye. The colorimetric LAMP assay provides on-and-off result of the LAMP reaction, not quantitative information. Since the quantification of the initial copy number of DNA is of importance for genetic diagnostics, we, for the first time, demonstrated how to quantify the DNA in the EBT mediated LAMP reaction. Due to the structral change of the EBT-Mg complexes, the color of the LAMP mixture is changed from violet to sky blue as the LAMP reaction proceeds. We noticed that the maxium absorption peak before and after the LAMP reaction is distinct, and accordingly shifted from 570 nm to 640 nm. We utilized the ratio between two particular absorbance (A(640)/A(570)) of EBT as an indicator for the LAMP process, and plotted it versus the reaction time. We used serially diluted DNA samples and produced quantitative calibration curves (the threshold time versus the initial copy number of DNA). Therefore, we could quantify the copy number of template DNAs in the EBT-mediated colorimetric LAMP reaction simply by measuring the two UV-vis absorption values at 570 nm and 640 nm.