Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR

Background The COVID-19 pandemic, caused by SARS-CoV-2, highlighted the need for accurate and scalable diagnostic tools such as RT-qPCR. However, false-negative results may occur due to viral mutations and RNA secondary structures within target regions. Methods High-performance computing (HPC) was used to compile SARS-CoV-2 genomic sequences from GenBank and GISAID and generate consensus sequences for primer and probe design. A region within the ORF8 gene was selected and evaluated alongside targets from the E and N genes and the RNase P control. Nasopharyngeal swab samples were collected from patients with a prior clinical diagnosis consistent with SARS-CoV-2 infection, as well as from volunteers, and total RNA was extracted using the MagMAX kit. RT-qPCR assays were performed in both single and multiplex formats. Denaturing solutions composed of tetraethylammonium chloride (TEA) and dimethyl sulfoxide (DMSO), as well as adjusted dNTP proportions based on viral nucleotide composition, were evaluated. Exploratory Ct-based performance metrics were estimated using predefined threshold criteria with the Caret package in R. Results A total of 126,576 SARS-CoV-2 genomes collected between January and December 2020 were used to construct a reference dataset. A target region within the ORF8 gene exhibiting predicted secondary structures was selected for primer and probe design. Forty-nine clinical samples were analyzed, of which 22 tested positive across the evaluated gene targets. Variability in detection patterns was observed across sampling periods. The evaluated formulations were associated with changes in Ct values in both single and multiplex RT-qPCR assays, depending on the conditions and sample set. Conclusions The incorporation of denaturing solutions and the adjustment of nucleotide proportions were associated with changes in RT-qPCR performance under the evaluated experimental conditions. These findings suggest that RNA secondary structure and nucleotide composition may influence assay behavior; however, further studies are required to assess the broader applicability of this approach.