Figure 1. Representative end-point electrophoresis showing RT-LAMP products after 1 hour reaction at temperatures of 60°C, 63°C, or 65°C using the SARS-N primer set. S1 and S2 represent isolated RNA from positive SARS-CoV-2 samples; N1 corresponds to isolated viral RNA from hCoV-229E as a negative control. A non-template control (NTC) was also included.
Specific amplification was observed using the SARS-N primers (Figure 1) or the SARS-E primers (Figure S1) at 65ºC in a panel of 6 positive and four negative samples. In contrast, nonspecific amplification of RNA from hCoV-229E at either 60ºC or 63ºC was observed (Figure 1). To optimize the RT-LAMP reaction, the addition of dUTP/UDG was assayed. Since this enzyme effectively catalyzes the removal of any uracil nucleotides that may be present prior to the amplification reaction, it would prevent carryover contamination during the RT-LAMP reaction. The results obtained here-in validate the effectiveness of this enzyme, as no nonspecific amplification was observed under any of the tested conditions (Figure S2)
Subsequently, to establish the optimal time conditions, end-point RT-LAMP was performed at 65ºC for 15, 30, 45, or 60 minutes, maintaining an equivalent concentration of viral RNA as template. After evaluating the fluorescence intensity of LAMP products, we observed that the reaction yield was the highest after 30 minutes or more (Figure S3). Consequently, an optimal reaction time of 30 minutes was set for RT-LAMP assays aimed at analyzing SARS-CoV-2. Besides, further optimization of primer concentration ranging from 0.025 to 2 µM each was also tested. According to our results, outer FIP/BIP primers at 1.6 µM, F3/B3 primers at 0.2 µM, and loop LF/LB primers at 0.4 µM exhibited optimal performance in LAMP reactions (Figure S2)