Table 1.
Primers, linearity and melting curve parameters for SARS-CoV-2 and human RNAse P amplification.
Fig 1.
HRM-RTqPCR assays for SARS-CoV-2 E, N and RdRp targets.
A. Derivative HRM curves for SARS-CoV-2 E (green), N (yellow) and RdRp (blue) and Human RNAse P (red) targets. The vertical blue lines divide the Pre-melt, Melt and Post-melt regions. B. Normalized HRM curves for SARS-CoV-2 E (green), N (yellow) and RdRp (blue) and Human RNAse P (red) targets. C. Dynamic extension (linearity) for the SARS-CoV-2 E (green), N (yellow) and RdRp (blue) gene target amplification. D. Normalized HRM curves for SARS-CoV-2 N region from COVID-19 clinical samples. E. Normalized HRM curves for SARS-CoV-2 RdRp region from COVID-19 clinical samples. F. Normalized HRM curves for SARS-CoV-2 E region from COVID-19 clinical samples.
Table 2.
Precision of HRM-RTqPCR assays targeting SARS-CoV-2 E, N and RdRp.
SARS-CoV-2 templates were assayed in 40 technical replicates, at 12, 10 and 8 copies/μL.
Fig 2.
Reproducibility of HRM-RTqPCR assays targeting SARS-CoV-2 E, N and RdRp.
SARS-CoV-2 templates were assayed in 40 technical replicates, at 12, 10 and 8 copies/μL.
Fig 3.
Boxplot of RNAse P Ct values between RT-qPCR TaqMan and RT-qPCR HRM assays and between swab, serum and saliva samples.
The Ct values for the amplification of the internal control, human RNAse P, between TaqMan and HRM assays were compared between all the collected clinical specimens (A) or between swab, serum and saliva samples (B). The outliers are represented as the black circles. In A, p = 0.634 (Mann-Whitney Rank Sum Test). In B, *P<0.05 (One-way ANOVA) and p = 1.00 (Swab x Saliva—Mann-Whitney Rank Sum Test).
Table 3.
Clinical validation of RT-qPCR assays targeting SARS-CoV-2 E, N and RdRp.
The parameters of clinical validation were calculated considering RT-qPCR with TaqMan assays as gold standard, using the OpenEpi (v. 3) software.
Fig 4.
Bland-Altman plots resulting from the pairwise comparison of Ct values between RT-qPCR TaqMan and HRM-RTqPCR assays targeting SARS-CoV-2 N, RdRp and E.
A. TaqMan N x HRM N targets. B. TaqMan N x HRM RdRp targets. C. TaqMan N x HRM E targets. Level of agreement between two assays, also known as observed mean difference (blue line at center), is presented as a function of the average Ct values of the two assays. Upper and lower limits of agreement (Upper and lower blue lines, respectively) defined by mean Ct difference ± 1.96 Standard Deviation (SD). The bias observed were 0.56, -1.71 and -0.89 to A, B and C, respectively.