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https://www.arca.fiocruz.br/handle/icict/63427
IN SILICO THERMODYNAMIC EVALUATION OF THE EFFECTIVENESS OF RT-LAMP PRIMERS FOR SARS-COV-2 VARIANTS DETECTION.
Diagnosis
LAMP primer design
SARS-CoV-2
DNA thermodynamic models
Melting temperature calculations
Affilliation
Universidade Federal de Minas Gerais. Departamento de Fı́sica. Belo Horizonte, MG, Brazil/Universidade Federal de Minas Gerais. Programa Interunidades de Pós-Graduação em Bioinformática. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Tecnologia em Vacinas, Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Universidade Federal de Minas Gerais. Departamento de Fı́sica. Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil/Fundação Oswaldo Cruz. Centro de Tecnologia em Vacinas, Belo Horizonte, MG, Brazil
Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, Brazil
Universidade Federal de Minas Gerais. Departamento de Fı́sica. Belo Horizonte, MG, Brazil
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.
Keywords
DNA mismatchesDiagnosis
LAMP primer design
SARS-CoV-2
DNA thermodynamic models
Melting temperature calculations
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