ArticleMolecular and cellular probes2021
Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes.
Article in Molecular and cellular probes, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Mesoscopic Calculation of Single Mismatches in RNA/DNA Hybrids: Strong Hydrogen Bonds of dTrG Affect CRISPR Off-Target Binding.The journal of physical chemistry. B · 2026Article
- Using machine learning models to predict the impact of template mismatches on polymerase chain reaction assay performance.Scientific reports · 2025Article
- OLTA: Optimizing bait seLection for TArgeted sequencing.Bioinformatics (Oxford, England) · 2025Article
- Reality check: testing theFrontiers in cellular and infection microbiology · 2025Article
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- A two-step process for in silico screening to assess the performance of qRTPCR kits against variant strains of SARS-CoV-2.BMC genomics · 2022Article
- Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR.Applied microbiology and biotechnology · 2022Article
- Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.Frontiers in microbiology · 2021Article
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Authors and funding
2 authors.
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Abstract
backgroundDNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primer or probe regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed.
methodsWe calculate the hybridization temperatures of primer/probe sets after aligning to SARS-CoV-2, SARS-CoV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5
resultsWe obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes.
conclusionsSome primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.
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