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 (A₆₄₀/A₅₇₀) 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.

中文翻译：

---

比色环介导的等温扩增过程的定量

我们提出了一种基于荧光铬黑T（EBT）染料的紫外可见光谱吸收度偏移的比色环介导的等温扩增（LAMP）的新型定量测定方法。比色LAMP测定提供了LAMP反应的开和关结果，而不是定量信息。由于DNA初始拷贝数的定量对于遗传诊断非常重要，因此我们首次展示了如何在EBT介导的LAMP反应中定量DNA。由于EBT-Mg配合物的结构变化，随着LAMP反应的进行，LAMP混合物的颜色从紫色变为天蓝色。我们注意到，LAMP反应之前和之后的最大吸收峰是明显的，因此从570 nm移至640 nm。我们利用了两个特定吸光度之间的比率（AEBT ₆₄₀ / A ₅₇₀）作为LAMP工艺的指标，并将其与反应时间作图。我们使用了系列稀释的DNA样品并产生了定量校准曲线（阈值时间与DNA的初始拷贝数）。因此，我们可以简单地通过测量570 nm和640 nm处的两个UV-vis吸收值来量化EBT介导的比色LAMP反应中模板DNA的拷贝数。