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February 2022
StephaniePoppinga RichardMolenkampa Joachim D.Weigelb Pim G.N.J.Mutsaersc Jolanda C.Voermansa Sandervan Boheemena Marc C.Shamiera Reina S.Sikkemaa David F.Nieuwenhuijsea Bas B.Oude Munninka Marion P.G.Koopmansa Jeroen J.A.van Kampena
Highlights
•SARS-CoV-2 variants can result in PCR target failure shown by CT-value discrepancy.
•Multiple targets are key to provide and maintain reliable molecular diagnostics.
•Genomic SARS-CoV-2 surveillance must also include primer and probe targeting areas.
•Public release of primer/probe sequences are crucial to maintain PCR quality.
Abstract
Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches.
StephaniePoppinga RichardMolenkampa Joachim D.Weigelb Pim G.N.J.Mutsaersc Jolanda C.Voermansa Sandervan Boheemena Marc C.Shamiera Reina S.Sikkemaa David F.Nieuwenhuijsea Bas B.Oude Munninka Marion P.G.Koopmansa Jeroen J.A.van Kampena
Highlights
•SARS-CoV-2 variants can result in PCR target failure shown by CT-value discrepancy.
•Multiple targets are key to provide and maintain reliable molecular diagnostics.
•Genomic SARS-CoV-2 surveillance must also include primer and probe targeting areas.
•Public release of primer/probe sequences are crucial to maintain PCR quality.
Abstract
Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches.
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