The impact of heat inactivation on RT-qPCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): an experience from the University Clinical Centre of Vojvodina, Serbia

Abstract

Handling clinical samples from patients suspected of SARS-CoV-2 infection puts healthcare workers at risk of exposure to infectious particles. To reduce this risk, samples are often heat-inactivated before nucleic acid isolation, but this procedure may affect the analytical sensitivity of the test. The aim of this study was therefore to evaluate the effects of heat inactivation (56 °C for 30 min) on RT-qPCR results of samples taken from nasopharyngeal and oropharyngeal (NP/OP) swabs collected from 200 symptomatic patients. Each sample was split into two aliquots - one subjected to heat inactivation and the other stored at 4 °C - followed by nucleic acid isolation and RT-qPCR analysis using the GeneFinder COVID-19 nucleic acid test. Heat inactivation did not significantly affect the overall SARS-CoV-2 detection rate (55.5 % vs. 55.0 % in untreated and heat-treated groups; χ²=0.01; p=0.91). However, discrepancies occurred in 15.3 % of samples, all with quantification cycle (Cq) values >31, including target loss, gain, or complete signal disappearance after heat treatment. Heat inactivation also slightly decreased Cq values for the RNA-dependent RNA polymerase (RdRp) and envelope (E) genes and increased those for the nucleocapsid (N) gene, with significant changes in strongly positive samples (Cq≤33). In positive samples (Cq≤40), the human ribonuclease (RNase) P gene also exhibited significantly higher Cq values after heat treatment. In the strongly positive subgroup, correlation analysis showed moderate correlation for RdRp and very strong correlation for the N and E genes, and a weaker correlation for weakly positive samples. In conclusion, heat inactivation at 56 °C for 30 min does not significantly affect viral gene detection but may diminish it in samples with low viral load.