Validation of qRT-LAMP assays for SARS-CoV-2 detection
Sample validation was carried out by qRT-LAMP with end-point melt curve step to verify the specificity according to primer annealing temperature. To establish the suitability of N and E genes as detection targets for SARS-CoV-2, a preliminary sensitivity test was performed on purified viral RNA from confirmed 50 positive and 10 negative samples. The results demonstrated that out of the 50 positive samples examined, 48 were detected using the N-gene primer set, and 43 were detected using the E-gene primer one. No false positives were obseved with either primer set. Indeed, they show a 100% specificity, indicating their high accuracy in identifying SARS-CoV-2. These results indicate that N-gene primer set is the most appropriate for SARS-CoV-2 detection by RT-LAMP.
To further assess the specificity and sensitivity of the N-gene primer set, we conducted a comparative analysis of a panel of 140 different pharyngeal swabs using qRT-PCR and qRT-LAMP methods (Figure 2). This primer set successfully detected viral RNA with a qRT-PCR cycle threshold (CT) of up to 30, with a sensitivity of 97.22% (70/72 positive samples) and 100% specificity of (68/48 negative samples). Notably, our results showed that qRT-LAMP using SARS-N primers was sensitive enough to detect viral RNA after just 30 min reaction (Figure 2A). Indeed, its limit of detection (LOD) was about 50 copies per reaction (equivalent to qPCR CT = 30 according to manufacturer specifications). Moreover, we observed a nice correlation between the RNA copies and the reaction time, revealing that 400 RNA copies per reaction can be detected as early as 13 minutes. Considering that retro-transcription step lasts for 15 minutes in qRT-PCR based methods, we found that qRT-LAMP was faster as amplification were actually evident even 11 minutes earlier.