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)