Fig. 4.

Development of the composite lavender green (LG) pH-sensitive indicator for IC-RT-LAMP-LG. A The lavender green indicator is a combination of two pH-sensitive indicators, XO (XO) and malachite green (MG). The determination of the optimal balance between the two dye components was performed through the iteration of xylenol orange concentration while the working concentration of MG was fixed at 0.02 mM in the RT-LAMP reaction that targets human 18 S rRNA. The stoichiometric ratio of dyes at 0.02 mM MG: 0.12 mM XO offers the clearest visual contrast between positive and negative results and the highest limit of detection (LoD) at 10 pg total RNA by which the incorporation of XO did not affect the assay's intrinsic LoD. B Corresponding AGE confirmation of the colorimetric results. C UV–Vis analysis of the positive and negative IC-RT-LAMP-LG reactions across stoichiometric variations of MG and XO where the positive reaction is characterized by the presence of the absorption peak at 620 nm that corresponds to the blue shade of MG in sub-neutral pH environment. Each line represents the average absorption of samples within the positive and negative groups. Lanes 1, 2, 3, 4, 5, 6, M and N: 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, molecular marker and negative control (template-free reaction), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)