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Validation and advantages of using novel RT-qPCR melting curve analysis assays for the identification of SARS-CoV-2 variants.
Juul, Sebastian; Spiegelhauer, Malene Roed; Petersen, Mette Neve; Flugt, Katharina Kirkegaard; Hansen, Nikolaj Vestergaard; Larsen, Helene; Jensen, Per Bo; Christensen, Ulf Bech; Petersen, Rasmus Koefoed; Friis-Hansen, Lennart.
  • Juul S; Research & Development, Pentabase A/S, Petersmindevej 1A, 5000, Odense C, Denmark. Sej@pentabase.com.
  • Spiegelhauer MR; Department of Clinical Biochemistry, Copenhagen University Hospital, Bispebjerg, Denmark.
  • Petersen MN; Department of Clinical Biochemistry, Copenhagen University Hospital, Bispebjerg, Denmark.
  • Flugt KK; Research & Development, Pentabase A/S, Petersmindevej 1A, 5000, Odense C, Denmark.
  • Hansen NV; Research & Development, Pentabase A/S, Petersmindevej 1A, 5000, Odense C, Denmark.
  • Larsen H; Center for Diagnostics, DTU Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark.
  • Jensen PB; Department of Clinical Biochemistry, Copenhagen University Hospital, Bispebjerg, Denmark.
  • Christensen UB; Research & Development, Pentabase A/S, Petersmindevej 1A, 5000, Odense C, Denmark.
  • Petersen RK; Research & Development, Pentabase A/S, Petersmindevej 1A, 5000, Odense C, Denmark.
  • Friis-Hansen L; Department of Clinical Biochemistry, Copenhagen University Hospital, Bispebjerg, Denmark.
Sci Rep ; 12(1): 13069, 2022 07 29.
Article in English | MEDLINE | ID: covidwho-1967624
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Cohort study / Diagnostic study / Observational study / Prognostic study Topics: Variants Limits: Humans Language: English Journal: Sci Rep Year: 2022 Document Type: Article Affiliation country: S41598-022-17339-0

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Cohort study / Diagnostic study / Observational study / Prognostic study Topics: Variants Limits: Humans Language: English Journal: Sci Rep Year: 2022 Document Type: Article Affiliation country: S41598-022-17339-0