{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown"],"volume":["299"],"submitter":["Zimmermann F"],"funding":["German Federal Ministry of Defence"],"pubmed_abstract":["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."],"journal":["Journal of virological methods"],"pagination":["114352"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC8570391"],"repository":["biostudies-literature"],"pubmed_title":["In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR."],"pmcid":["PMC8570391"],"pubmed_authors":["Urban M","Zwirglmaier K","Zimmermann F","Kruger C","Wolfel R","Walter M"],"additional_accession":[]},"is_claimable":false,"name":"In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR.","description":"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.","dates":{"release":"2022-01-01T00:00:00Z","publication":"2022 Jan","modification":"2025-04-18T17:27:34.098Z","creation":"2022-02-11T13:01:03.561Z"},"accession":"S-EPMC8570391","cross_references":{"pubmed":["34748815"],"doi":["10.1016/j.jviromet.2021.114352"]}}