Mesoscopic Evaluation of DNA Mismatches in PCR Primer-Target Hybridisation to Detect SARS-CoV-2 Variants of Concern

Mismatches are any type of base-pairs other than AT and CG. They are an expected occurrence in PCR primer-target hybridisation and may interfere with the amplification and in some cases even prevent the detection of viruses and other types of target. Given the natural occurrence of mutations it is expected that the number of primer-target mismatches increases which may result in a larger number of false-negative PCR diagnostics. However, mismatches may equally improve the primer-target hybridisation since some types of mismatches may stabilize the helix. Only very recently have thermodynamic parameters become available that would allow the prediction of mismatch effects at buffer conditions similar to that of PCR. Here we collected primers from WHO recommendation and aligned them to the genomes of the current variants of concern (VOC): Alpha, Beta, Gamma and Delta variants. We calculated the hybridisation temperatures taking into account up to three consecutive mismatches with the new parameters. We assumed that hybridisation temperatures to mismatched alignments within a range of 5 ∘ C of the non-mismatched temperature to still result in functional primers. In addition, we calculated strict and partial coverages for complete and mismatched alignments considering only single, double and triple consecutive mismatches. We found that if mismatches are taken into account, the coverage of WHO primers actually increase for VOCs and for the Delta variant it becomes 100%. This suggest that, at least for the moment, these primers should continue to be effective for the detection of VOCs. © 2021, Springer Nature Switzerland AG.

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes.

BACKGROUND: DNA 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. METHODS: We 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 ∘C to the fully matched reference temperature. RESULTS: We 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. CONCLUSIONS: Some 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.