Evaluating the Effect of Storage Temperature, Duration, and Multiple Freeze–Thaw Cycles on the RT-PCR Detection of SARS-CoV-2 RNA in Patient Samples

Abstract

Background: Real-time reverse transcription polymerase chain reaction (RT-PCR) is currently the standard method for detecting SARS-CoV-2 infection. Nevertheless, preanalytical issues such as storage temperature, exposure time and repeated freeze–thawing cycles can affect RNA integrity and reduce diagnostic sensitivity.

Objective: The purpose of this study was to investigate the influence of various storage temperatures, storage times, multiple freeze–thaw cycles on the stability and RT-PCR detectability of SARS-CoV-2 in clinical specimens.

Methods: A pool of nasopharyngeal and oropharyngeal swab samples obtained from SARS-CoV-2–positive patients in VTM was divided into aliquots, each submitted for storage on day 1 at different temperatures (room temperature), 4°C, −20°Cand −80°C) over time (1, 2, 3, 5 and12 days). Aliquots were frozen and thawed for various times. The viral RNA was extracted with an automatic extractor and amplified by RT-PCR to the RdRp gene. Alterations in cycle threshold (Ct) were measured.

Results: Excellent RNA stability was shown by samples kept at −80°C, with little variation in Ct values at all time periods. For up to 72 hours, storage at 4°C was acceptable; however, longer storage led to a progressive increase in Ct. Significant RNA degradation occurred after 48–72 hours of room temperature storage, especially in samples with low virus loads. Ct values increased gradually with repeated freeze-thaw cycles, and after three or more cycles, there was a noticeable loss of detectability.

Conclusion: Preanalytical storage conditions have a considerable impact on SARS-CoV-2 RT-PCR results. Maintaining RNA integrity and ensuring accurate molecular diagnosis need optimal storage at −80°C and minimizing freeze-thaw cycles.