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. 2022 Feb:300:114397.
doi: 10.1016/j.jviromet.2021.114397. Epub 2021 Dec 1.

Diminished amplification of SARS-CoV-2 ORF1ab in a commercial dual-target qRT-PCR diagnostic assay

Stephanie Popping  1 , Richard Molenkamp  2 , Joachim D Weigel  3 , Pim G N J Mutsaers  4 , Jolanda C Voermans  2 , Sander van Boheemen  2 , Marc C Shamier  2 , Reina S Sikkema  2 , David F Nieuwenhuijse  2 , Bas B Oude Munnink  2 , Marion P G Koopmans  2 , Jeroen J A van Kampen  5
Affiliations

Diminished amplification of SARS-CoV-2 ORF1ab in a commercial dual-target qRT-PCR diagnostic assay

Stephanie Popping et al. J Virol Methods. 2022 Feb.

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.

Keywords: Cobas; Dual-target assay; Molecular diagnostics SARS-CoV-2; ORF1ab mutations; PCR amplification; SARS-CoV-2.

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Conflict of interest statement

Sequencing was funded by ZonMW (grant agreement no: 10150062010005).

SP: received financial support from Gilead Sciences outside of the submitted work.

RM; RS; MK; JW; PM; BOM; DN; JvV; MS; SvB; Jvk: reported no conflict of interest.

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Supplementary concepts