PT  - JOURNAL ARTICLE
AU  - Fee Zimmermann
AU  - Maria Urban
AU  - Christian Krüger
AU  - Mathias Walter
AU  - Roman Wölfel
AU  - Katrin Zwirglmaier
TI  - In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR
AID  - 10.1101/2021.06.07.447338
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.06.07.447338
4099  - http://biorxiv.org/content/early/2021/06/07/2021.06.07.447338.short
4100  - http://biorxiv.org/content/early/2021/06/07/2021.06.07.447338.full
AB  - A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases – as seen with the B.1.1.7 variant – can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from −0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charité RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.Competing Interest StatementThe authors have declared no competing interest.