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

Sebastian Juul, Malene Roed Spiegelhauer, Mette Neve Petersen, Katharina Kirkegaard Flugt, Nikolaj Vestergaard Hansen, Helene Larsen, Per Bo Jensen, Ulf Bech Christensen, Rasmus Koefoed Petersen, Lennart Friis-Hansen


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.

Original languageEnglish
Article number13069
JournalScientific Reports
Issue number1
Pages (from-to)1-12
Number of pages12
Publication statusPublished - 29 Jul 2022


  • COVID-19/diagnosis
  • Humans
  • Mutation
  • Reverse Transcription
  • SARS-CoV-2/genetics
  • Sensitivity and Specificity


Dive into the research topics of 'Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants'. Together they form a unique fingerprint.

Cite this