Reverse transcription-quantitative PCR assays for detecting SARS-CoV-2 using subgenomic RNA load

SARS-CoV-2, the virus responsible for COVID-19, triggers the synthesis of full-length genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) in host cells upon infection. Traditional PCR aimed at targeting gRNA to detect viral presence is insufficient. sgRNAs serve as novel markers for active viral replication. However, the utility of reverse transcription-quantitative PCR (RT-qPCR) assays targeting sgRNAs as indicators of active viral load and infectivity in rodent models has not been validated. We developed four RT-qPCR assays targeting the SARS-CoV-2 genes-ORF1ab, N, E, and E-sgRNA and two RT-qPCR assays for housekeeping genes Hamster <i>G</i> <i>apdh</i>, and Mouse <i>Actb</i>. We used serial dilutions to establish standard curves for quantification. These assays demonstrated high amplification efficiency (96%-97 %) and a strong correlation between the cycle threshold (Ct) values and logarithmic copy number of the genes (R² = 0.9933-0.9996). Analyzing 102 residual rodent lung tissue samples, we compared the viral loads quantified using RT-qPCR assays with those determined by viral culture. A strong correlation emerged between the RT-qPCR assays' detection of positivity and the viral culture results. Notably, the quantification of viral loads using the E-sgRNA RT-qPCR assay correlated more closely with viral culture outcomes than with other targets (r = 0.93, p < 0.001). These results underscore the sgRNA RT-qPCR assay's potential for tracking actively replicating viruses in rodent models infected with SARS-CoV-2, offering a reliable alternative to traditional viral culture methods.