Ethanol improves RNase P stability in nasopharyngeal samples stored at room temperature for SARS-CoV-2 RT-qPCR diagnosis.

Despite the end of the global health emergency, COVID-19 and its etiological agent, SARS-CoV-2, remain topics of high scientific and public health relevance due to the continued circulation of the virus, the emergence of new variants, and the persistent risk of outbreaks. RT-qPCR is the gold standard procedure for SARS-CoV-2 detection, but its reliability depends on preserving viral RNA and the human Ribonuclease P (RNase P; internal control) target during sample storage and transport. This study aimed to evaluate the effect of different preservation conditions on nucleic acid stability for SARS-CoV-2 detection by RT-qPCR: samples maintained in 0.9% saline and samples supplemented with absolute ethanol to reach a final concentration of 70% (v/v), under different storage temperature conditions. Nasopharyngeal swab samples, initially collected and transported in 0.9% saline and previously classified into different viral load categories, were pooled and redistributed into the experimental storage conditions: ethanol or saline under refrigerated conditions (4°C) or at room temperature. RNA was extracted using an automated magnetic bead-based method and analyzed by RT-qPCR targeting the SARS-CoV-2 E gene and the human RNase P internal control, with nucleic acid stability monitored over a 12-day storage period. Viral RNA detection remained stable in both storage media; however, RNase P detection progressively declined in saline, particularly during room temperature storage, while ethanol maintained consistent amplification. These findings indicate that 70% ethanol better maintains the stability of the human internal control (RNase P) than saline during room temperature storage of nasopharyngeal samples for up to 12 days.