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Background:  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.    Methods:  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.    Results:  These sequences were then used to create  ORF8 -specific primers and probes to validate single and multiplex RT-qPCR protocols both  in silico  and experimentally using genes  E  (Berlin protocol) and  N  (CDC protocol) as targets.    Conclusions:  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.

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