Figure 4. Representative validation of the RT-LAMP/DNA-nanoprobes detection system for SARS-CoV-2 (samples S18 to S23, left panels) or aMPV (samples M6 to M11, right panels). A) Representative digital pictures acquired after 0 min or 30 min incubation at 37°C of respective RT-LAMP products with either SARS-N- (left panels) or aMPV-F2-nanoprobes (right panels) are shown. Histograms represent their maximum absorbance intensity at λ= 540 nm at 30 min incubation. B) qRT-LAMP amplification plots after 45 min incubation at 65oC. Isolated RNA from hCoV-229E (N1) or from IBV (N2) was used as negative control for SARS-CoV-2 and aMPV, respectively.
The percentages of sensitivity and specificity obtained by coupling RT-LAMP to DNA-nanoprobes for molecular detection were calculated using a conventional confusion matrix (Table 1). It is worth noting that only two out of 72 positive SARS-CoV-2 were determined as false negatives (98.57% accuracy; 97.18% Matthews Correlation Coefficient (M.C.C.)). Regarding aMPV, detection values were slightly lower, since 4 out of 33 positive aMPV samples were determined as false negatives (92.00% accuracy; 84.34% M.C.C.)
In summary, we propose the following reaction conditions for both SARS-CoV-2 or aMPV detection: the RT-LAMP should carried out in presence of UDG, 20 U of RNAse inhibitor, primers FIP/BIP at 1.6 µM, F3/B3 at 0.2 µM, and LF/BF at 0.4 µM, using 5 µL of purified RNA for 25 µL reactions, amplification for 30 minutes at 65ºC, and heat inactivation for 5 min at 80ºC. , amplicons are visualized by adding 1,5 µL of DNA-nanoprobes in the adequate buffer and allowing the reaction to happen for 30 min at 37ºC.
Table 1. Performance parameters of the RT-LAMP/nanoprobe detection system for SARS-CoV-2 or aMPV (N.P.V, Negative Predictive Value; F.D.R, False Discovery Rate; F.P.R, False Positive Rate; F.N.R, False Negative Rate; M.C.C., Matthews Correlation Coefficient)