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

Sep 29, 2026

Uploaded by Ben

Healthcare workers face exposure risk when handling clinical samples suspected of SARS-CoV-2, so laboratories often apply heat inactivation before nucleic acid isolation. This study evaluates whether heat inactivation at 56 °C for 30 min alters RT-qPCR performance for nasopharyngeal and oropharyngeal swabs from 200 symptomatic patients. Samples were split into heat-treated and 4 °C stored aliquots for extraction and GeneFinder COVID-19 RT-qPCR testing. Overall detection was unchanged, but discrepancies occurred mainly in low viral-load samples.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages9

Reading Time23 min

AccessFree to download

Background

Study aim and design

Sample handling and heat-inactivation protocol

RT-qPCR testing approach

Results

Overall detection rate

Sample discrepancies and Cq changes

Interpretation and conclusion

Stojčević Maletić J, et al. The impact of heat inactivation on RT-qPCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Arh Hig Rada Toksikol 2025;76:242–250 242 Original article DOI: 10.2478/aiht-2025-76-3973 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 Jelena Stojčević Maletić1,2, Iva Barjaktarović1,2, Ljiljana Andrijević1, Katarina Bačulov1,2, Slobodanka Bogdanović Vasić3, and Diandra Pintać Šarac1 1 University of Novi Sad Faculty of Medicine, Novi Sad, Serbia 2 University Clinical Centre of Vojvodina, Novi Sad, Serbia 3 Šabac Academy of Vocational Studies, Šabac, Serbia [Received in March 2025; Similarity Check in March 2025; Accepted in October 2025] 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 RTqPCR 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. KEYWORDS: COVID-19; GeneFinder nucleic acid test; human RNaseP gene; nasopharyngeal swabs; oropharyngeal swabs; quantification cycle; real-time quantitative polymerase chain reaction; RNA isolation On 11 March 2020, the World Health Organization (WHO) declared the coronavirus disease 2019 (COVID-19) a global pandemic (1, 2). By June 2024, about 776 million cases of SARSCoV-2 infection and seven million deaths had been documented worldwide (3) . Laboratory COVID-19 testing was crucial for establishing the incidence of infection as precisely as possible, but due to the limited time window, the WHO forewent serological assays or the nucleic acid amplification tests and instead recommended the use of amplification of viral ribonucleic acid (RNA) by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) as the gold standard for diagnosis (4–9) . RT-qPCR can identify several genes encoding viral structural proteins, such as the spike (S), envelope (E), membrane (M), and nucleocapsid (N) (10), along with eight accessory proteins. It can also detect the open reading frame-1 (ORF1ab) gene, which encodes non-structural polyproteins which are cleaved to encode 16 nonstructural proteins (NSP1–16) responsible for viral transcription Corresponding author: Jelena Stojčević Maletić, University of Novi

FAQs

Why do laboratories use heat inactivation before RT-qPCR for SARS-CoV-2?

Heat inactivation reduces exposure risk to infectious particles during sample processing. It may, however, change nucleic acid quality and affect test analytical sensitivity.

What heat-inactivation conditions were evaluated in the study?

The study assessed heat inactivation at 56 °C for 30 minutes compared with aliquots stored at 4 °C before nucleic acid extraction and RT-qPCR analysis.

Did heat inactivation change SARS-CoV-2 detection overall?

Overall detection rates were not significantly different between untreated and heat-treated groups. Differences mainly appeared in a subset of samples with high Cq values, consistent with lower viral loads.

Sep 29, 2026

Uploaded by Ben