Introduction:

RT-qPCR performance to detect SARS-CoV-2 varies because SNPs at primer/probe sites and RNA secondary structures (notably 5'/3' UTR stem-loops) can hinder hybridization/polymerase elongation, altering Ct and fluorescence. A denaturant solution (DS: composed of Tetramethylammonium Chloride and Dimethyl Sulfoxide) can disrupt these structures and improves its detection.

Objective:

Evaluate the DS efficiency in RT-qPCR to SARS-CoV-2 detection from single and multiplex saliva samples in COVID-19 positive patients.

Methodology:

To establish the hybridization pattern, in silico analysis was carried out among primers and probes authorized by CDC (Centers for Disease Control and Prevention) with respect to the consensus region of SARS-CoV-2 N gen, and their secondary and tertiary structures were generated to establish variable regions. Individual and pooled RT-qPCR with DS reactions, were validated from total RNA from 20 of 40 saliva samples positive to SARS-CoV-2.

Results:

N1 consensus region of SARS-CoV-2 variants: Alfa, Beta, Delta, Gamma, GH490R Lambda, Mu y Ómicron from 2021, exhibited a high variation respect to N2 region, but were located in bubbles near to a stem and in the spacer region. Individual RT-qPCR to N1 and N2 regions showed no significant statistical differences in Cts, however, they were lower, with a remarkable difference in fluorescence signal, to N1 region in pooled RT-qPCR reactions.

Conclusion:

The procedure evidences the advantages in the inclusion of DS in RT-qPCR to SARS-CoV-2 detection in saliva samples.