RESEARCH ARTICLE
In silico Thermodynamic Evaluation of the Effectiveness of RT-LAMP Primers for SARS-CoV-2 Variants Detection
Pâmella Miranda1, 2, *, Pedro A. Alves3, 4, Rubens L. do Monte-Neto3, Gerald Weber1
Article Information
Identifiers and Pagination:
Year: 2024Volume: 4
E-location ID: e26669587279780
Publisher ID: e26669587279780
DOI: 10.2174/0126669587279780240130063422
Article History:
Received Date: 05/10/2023Revision Received Date: 22/12/2023
Acceptance Date: 16/01/2024
Electronic publication date: 13/02/2024
Collection year: 2024
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Background
Viral mutations are the primary cause of mismatches in primer-target hybridisation, affecting the sensibility of molecular techniques, and potentially leading to detection dropouts. Despite its importance, little is known about the quantitative effect of mismatches in primer-target hybridisation. We have used up-to-date and highly detailed thermodynamic model parameters of DNA mismatches to evaluate the sensibility to variants of SARS-CoV-2 RT-LAMP primers.
Methods
We aligned 18 RT-LAMP primer sets, which underwent clinical validation, to the genomes of the wild-type strain (ws), 7 variants and 4 subvariants, and calculated hybridisation temperatures allowing up to three consecutive mismatches. We calculated the coverage when the mismatched melting temperature fell by more than 5°C in comparison to the matched alignments. If no mismatches were considered, the average coverage found was 94% for ws, falling to the lowest value for Omicron, i.e., 84%.
Results
However, considering mismatches, the coverage was much higher, i.e., 97% (ws) to 88% (Omicron). Stabilizing mismatches (higher melting temperatures) accounted for roughly 1/3 of this increase. The number of primer dropouts increased for each new variant; however, the effect was much less severe if mismatches were considered.
Conclusion
We suggest using melting temperature calculations to continuously assess the trend of primer dropouts.
