Figure 3. Representative qRT-LAMP amplification plot using aMPV-F primer
set. RNAs isolated from aMPV-positive samples (M1 to M5) were analyzed.
SHS is a commercial live attenuated vaccine used as positive control. To
assess potential cross-reactivity, viral RNAs from positive samples of
IBV-, ILTV-infected animals, and live-attenuated vaccine for NDV were
used (referred as N2, N3, and N4, respectively)
Validation of DNA-nanoprobe detection system coupled to
RT-LAMP
To assess the usefulness of the DNA-nanoprobes as a colorimetric tool
for the molecular detection, the RT-LAMP products for SARS-CoV-2 (140
samples) or aMPV (50 samples) were incubated with the corresponding
SARS-N- or aMPV-F2-nanoprobes, for 90 minutes at 37ÂșC (Figure 4A, left
panels, and right panels). qRT-LAMP for SARS-CoV-2 or aMPV were carried
out in parallel to confirm RT-LAMP amplification (Figure 4B, lower
panels). During the incubation period, spectrophotometric measurements
at UV-Visible wavelengths were acquired at 10-minute intervals.
Significantly, we observed that a 30-minute incubation period proved
sufficient for identifying and distinguishing positive samples. In fact,
at this time, in presence of RT-LAMP products, the initially red-color
reaction persisted due to the stabilization of nanoprobe molecules upon
recognizing the target. Conversely, reactions turned colorless in the
absence of target due to nanoprobe destabilization in the incubation
buffer.