ArticleJournal of virological methods2022
In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR.
Article in Journal of virological methods, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 17 citations in OpenAlex.
- SARS-CoV-2 Evolution and Its Implications for RT-PCR Diagnostic Performance.Journal of medical virology · 2026Article
- Overcoming Target Drift: Development and Validation of a One-Step TaqMan qPCR Assay for Epidemiological Surveillance ofMicroorganisms · 2026Article
- Molecular Detection and Genetic Characterization of Avian Leukosis Virus From Field Outbreaks in Bangladesh.Veterinary medicine and science · 2024Article
- Enzyme-Free Exponential Amplification via Growth and Scission of Crisscross Ribbons from Single-Stranded DNA Components.Journal of the American Chemical Society · 2024Article
- The Mutational Landscape of SARS-CoV-2.International journal of molecular sciences · 2023Article
- Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2.Microorganisms · 2023Article
- Tailored Multiplex Real-Time RT-PCR with Species-Specific Internal Positive Controls for Detecting SARS-CoV-2 in Canine and Feline Clinical Samples.Animals : an open access journal from MDPI · 2023Article
- Identification of mutations in SARS-CoV-2 PCR primer regions.Scientific reports · 2022Article
- A Comparison of Pharyngeal Swabs and Tracheal Secretions for the Diagnosing of COVID-19.Biomedicines · 2022Article
- Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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