The Comparative Analysis of Two RT-qPCR Kits for Detecting SARS-CoV-2 Reveals a Higher Risk of False-Negative Diagnosis in Samples with High Quantification Cycles for Viral and Internal Genes

Figure 1

Determination of total RNA extracted from nasopharyngeal swab samples (NPSs) loading volume and the limit of detection (LoD) of SARS-CoV-2 probes using TAAG RT-qPCR kit. The upper section of the figure represents the Cq value paired comparison for total RNA extracted from NPSs loading volume determination. The analysis was made from the same NPSs using the manufacturer’s recommended volume of 5 μl and 2 μl of total RNA extracted. In the graphs, the comparison was made for (a) RNase P internal reference, (b) N1, and (c) E viral gene probes. Each spot is a different analyzed sample for each volume condition (2 μl; 5 μl). On (a-c), the mean ± standard deviation (mean ± SD) for all the samples evaluated is indicated for each total RNA volume condition (2 μl; 5 μl). The line linking the spots indicate the paired result obtained for the same sample assessed using the two different volume conditions. Samples with Cq = 46 denote no amplification (indicated in the graph by a black broken line). For statistical analysis, paired two-sided student' t-test was applied (n = 80 random NPSs, n = 31 N1 gene, and n = 31 E gene random positive SARS-CoV-2 NPSs, ). The lower section of the figure represents (d) the number of amplified samples using TAAG probes loading 2 and 5 μl of total RNA extracted. Limit of detection (LoD) for (e) N1 gene probe and (f) E gene probe for SARS-CoV-2 detection. The analysis included 10-fold serial dilutions from a reference pool made from randomized ten total RNA NPS-extracted samples with a Cq value close to 20 (previously obtained using the Thermo Fisher kit). For the LoD determination, a linear regression was performed.

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