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 2

Comparative analysis for the detection of SARS-CoV-2 from random-chosen nasopharyngeal swab samples (NPSs) using Thermo Fisher and TAAG RT-qPCR kits. The comparison was made from the same NPSs using the previously optimized volume of 2 μl of total RNA extracted. The upper section of the figure represents the analysis for the amplification of RNase P internal reference gene probes from 91 random NPSs. (a) Cq value for internal reference RNase P gene probe amplification using Thermo Fisher and TAAG RT-qPCR kits (n = 91 NPSs). In graph, the horizontal red-dotted line represents the LoD for RNase P reference gene probe (Cq = 36.9) by Thermo Fisher kit, and the horizontal blue-dotted line represents the maximum recommended Cq value (Cq = 38) by the manufacturer of suitable sample for diagnosis using TAAG RT-qPCR kit RNase P reference gene probe. Samples with Cq = 46 denote no amplification (indicated in the graph by a black broken line). (b) Summary of the percentage of suitable NPSs for diagnosis using the internal reference RNase P gene probe (detailed on (a)) by both RT-qPCR kits. (c) RFU value from the same samples analyzed on (a). Samples with a Cq value > 38 were considered as not suitable according to manufacturer’s recommendations. For statistical analysis, a paired two-sided student’s t-test was applied (n = 91 random NPSs, ). The lower section of the figure represents the analysis for the amplification of SARS-CoV-2 viral gene probes from 91 random NPSs. Paired comparison for (d) Cq and (e) RFU values from viral gene probe amplification using Thermo Fisher (ORF1ab, N, and S gene probes) and TAAG (N1 and E gene probes) kits (n = 20). In graph (d), the horizontal colored-dotted lines blue and green, respectively, represent the experimental LoD for N1 gene (Cq = 29.87) and E gene (Cq = 31.09) probes of TAAG RT-qPCR kit. The horizontal black dotted line represents the maximum Cq value (Cq = 37) recommended by the manufacturer for positive sample diagnosis using TAAG RT-qPCR viral probes. Samples with Cq = 46 denote no amplification (indicated in the graph by a black broken line). For statistical analysis, a paired one-way ANOVA-test with multiple comparison test analysis applied. Lowercase letters above each probe's spot columns indicate which probes do not show significant differences between them. (n = 20 NPSs, ^∗∗∗∗p < 0.05). In graphs (a), (c), (d), and (e), the mean ± standard deviation (mean ± SD) is indicated for the viral gene probe amplification obtained for all the samples evaluated. The line linking the spots indicated the paired result obtained for the same sample assessed by both RT-qPCR kits. (f) Summary of the number of positive NPSs detected using Thermo Fisher (ORF1ab, N, and S probes) and TAAG RT-qPCR kit (N1 and E viral gene probes applying brochure or experimental LoD criteria ((n) = 20). (g) Summary of the number of positive NPSs diagnosed using TAAG RT-qPCR kit considering the RNase P internal control amplification. In the graph, “(+) SARS-CoV-2” represents a positive virus diagnosis, and “(+) RNase P” represents the amplification of the internal reference gene. (h) Summary of the percentage of false negative NPSs diagnosed by TAAG RT-qPCR kits applying brochure or experimental LoD criteria.