Figure 2.

Optimization of the RT-LAMP assay. (A) Optimization of the ratio of outer primers to inner primers. Lanes 1–5 represent 1:1, 1:2, 1:4, 1:8, and 1:16, respectively. (B) Optimization of the concentration of loop primers. Lanes 1–5 represent 0.1, 0.2, 0.4, 0.6, and 0.8 μM, respectively. (C) Optimization of the concentration of dNTPs. Lanes 1–5 represent 0.8, 1, 1.2, 1.4, and 1.6 mM, respectively. (D) Optimization of the concentration of MgSO₄. Lanes 1–5 represent 2, 4, 6, 8, and 10 mM, respectively. (E) Optimization of reaction time. Lanes 1–5 represent 30, 40, 50, 60, and 70 min, respectively. (F) Optimization of reaction temperature. Lanes 1–5 represent 59, 61, 63, 65, and 67°C, respectively. In each figure, Lane 6 represents the negative control and the red asterisk indicates the optimal conditions for this assay. Each reaction was performed with three repeats, and one representative readout is shown in the figures. The grayscale value of each agarose gel electrophoretic band was measured by ImageJ software (National Institutes of Health).