TY - JOUR
T1 - Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants
AU - Juul, Sebastian
AU - Spiegelhauer, Malene Roed
AU - Petersen, Mette Neve
AU - Flugt, Katharina Kirkegaard
AU - Hansen, Nikolaj Vestergaard
AU - Larsen, Helene
AU - Jensen, Per Bo
AU - Christensen, Ulf Bech
AU - Petersen, Rasmus Koefoed
AU - Friis-Hansen, Lennart
N1 - Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - 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.
AB - 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.
KW - COVID-19/diagnosis
KW - Humans
KW - Mutation
KW - Reverse Transcription
KW - SARS-CoV-2/genetics
KW - Sensitivity and Specificity
U2 - 10.1038/s41598-022-17339-0
DO - 10.1038/s41598-022-17339-0
M3 - Journal article
C2 - 35906388
AN - SCOPUS:85135140166
SN - 2045-2322
VL - 12
JO - Scientific Reports
JF - Scientific Reports
M1 - 13069
ER -