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.