Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants

Abstract

Reverse transcription quantitative PCR (RT-qPCR) assays are gold standard in diagnosing SARS-CoV-2 infection and play a major role in viral subtyping for rapid detection and monitoring of important mutations, containing the spread of new virus variants. We wanted to compare RT-qPCR melting curve analysis assays to Sanger Sequencing for detection of variants within the SARS-CoV-2 spike glycoprotein and examined their sensitivity and specificity. Samples positive for SARS-CoV-2 (n = 663 + 82) were subtyped using both Sanger sequencing and five RT-qPCR melting curve analysis assays specific for the mutations N501Y, P681H, E484K, K417N/T, and N439K. The results of the two methods were compared. The training cohort and the clinical validation cohort showed equally, or significantly better sensitivity of the assays compared to the Sanger sequencing. The agreement of the Sanger sequencing and the assays ranged from 92.6 to 100% for the training cohort and 99.4-100% for the clinical validation. The sensitivity, specificity, and turn-around time of the RT-qPCR melting curve analysis assays are well-suited for clinical monitoring of VOCs, making the assays an important tool in contact tracing and risk stratification. Furthermore, the assays were able to indicate the presence of new mutations in the complementary sequence to the mutation-specific probes.

Figure 1

Created with BioRender.com The binding of the wild type and mutation sequence to the mutation specific probe. The probe has higher affinity for the mutation sequence and will result in a higher melting point temperature (Tm). The probe has a lowered affinity for the WT sequence due to the single nucleotide mismatch resulting in a decreased Tm.

Figure 2

The melting curve analysis of the K417N mutation combined with a WT (K417), K417N and K417T complementary strand. The assay is specific for the K417N mutation resulting in the highest affinity for the sequence encoding Asparagine (N) in codon 417 and a Tm of 64.4 °C. The assay has the additional function as it can detect the K417T mutation at Tm 55.66 °C as well.

Figure 3

Five samples for the P.1 + P681H, B.1.351, B.1.1.7, B.1.258 and WT were analyzed. The melting curve of the five samples are illustrated for the N439K assay. As it is shown in the graph the N439K mutation can be distinguished by approximately 5 °C from the WT sequence.

Figure 4

Five samples for the P.1 + P681H, B.1.351, B.1.1.7, B.1.258 and WT were analyzed. The melting curve of the five samples are illustrated for the N501Y assay. As it is shown in the graph the N501Y mutation can be distinguished by approximately 6 °C from the WT sequence.

Figure 5

Five samples for the P.1 + P681H, B.1.351, B.1.1.7, B.1.258 and WT were analyzed. The melting curve of the five samples are illustrated for the P681H assay. As it is shown in the graph the P681H mutation can be distinguished by approximately 10 °C from the WT sequence.

Figure 6

Five samples for the P.1 + P681H, B.1.351, B.1.1.7, B.1.258 and WT were analyzed. The melting curve of the five samples are illustrated for the E484K assay. As it is shown in the graph the E484K mutation can be distinguished by approximately 7 °C from the WT sequence.

Figure 7

Five samples for the P.1 + P681H, B.1.351, B.1.1.7, B.1.258 and WT were analyzed. The melting curve of the five samples are illustrated for the K417N/T assay. As it is shown in the graph the K417N mutation can be distinguished by approximately 4 °C from the WT sequence and the K417T mutation can be distinguished 4 °C below the WT sequence.