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 3

Comparative analysis between Thermo Fisher and TAAG RT-qPCR kits with a known Cq value close to 30 (for ORF1ab gene probe, Thermo Fisher). The comparison was made for 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 RNase P internal reference gene probe amplification from 10 NPSs previously diagnosed as SARS-CoV-2 positive by the Thermo Fisher RT-qPCR kit with a Cq value close to 30. (a) Paired comparison of Cq value of RNase P internal reference gene probes from Thermo Fisher and TAAG kits. 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 for suitable sample 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). For statistical analysis, a paired two-sided student's t-test was applied (n = 10 NPSs, ). (b) Number of suitable samples diagnosed with a Cq value within the detection range of the internal reference gene probes. The lower section of the figure represents the analysis for the viral gene probe amplification from 10 NPSs previously diagnosed as SARS-CoV-2 positive by the Thermo Fisher RT-qPCR kit with a Cq value close to 30. (c) Paired comparison of Cq value of viral gene probes from Thermo Fisher (ORF1ab viral gene probe) and TAAG (N1 and E viral gene probes) kits. In graph, the horizontal colored-dotted lines blue, green, and red, respectively, represent the experimental LoD for N1 gene (Cq = 29.87), E gene (Cq = 31.09), and ORF1ab gene (Cq = 37.14) viral gene probes. Samples with Cq = 46 denote no amplification (indicated in the graph by a black broken line). For statistical analysis, paired one-way ANOVA-test with multiple comparison test analysis was applied. (n = 10 NPSs, ). In graphs (a) and (c), 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 indicate the paired result obtained for the same sample assessed by both RT-qPCR kits. (d) 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. (e) Summary of the percentage of false negative NPSs diagnosed by TAAG RT-qPCR kits applying brochure or experimental LoD criteria.