<ns6:p><ns6:bold>Background:</ns6:bold> Recent estimates indicate that the COVID-19 pandemic, which is caused by the SARS-CoV-2 virus, could be effectively controlled via the development and implementation of diagnostic tools such as quantitative reverse transcription PCR (RT-qPCR). However, this reaction often generates false-negative results due to novel mutations and can also be affected by the secondary structure of the RNA transcripts that derive from the gene sequence used for diagnostic purposes.</ns6:p><ns6:p> <ns6:bold>Methods:</ns6:bold> Using high-performance computing, we consolidated a global SARS-CoV-2 genome repository encompassing 19,317 genomes from the GenBank database and 107,259 from the GISAID database to generate monthly SARS-CoV-2 consensus sequences from January to December 2020.</ns6:p><ns6:p> <ns6:bold>Results:</ns6:bold> These sequences were then used to create <ns6:italic>ORF8</ns6:italic>-specific primers and probes to validate single and multiplex RT-qPCR protocols both <ns6:italic>in silico</ns6:italic> and experimentally using genes <ns6:italic>E</ns6:italic> (Berlin protocol) and <ns6:italic>N</ns6:italic> (CDC protocol) as targets.</ns6:p><ns6:p> <ns6:bold>Conclusions:</ns6:bold> Our findings demonstrated that RT-qPCR Ct values were improved by the inclusion of either a denaturing solution composed of tetraethylammonium chloride (TEA) and dimethyl sulfoxide (DMSO) and by adjusting nucleotide proportions based on the SARS-CoV-2 genome.</ns6:p>