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.