Prolonged Unfrozen Storage and Repeated Freeze-Thawing of SARS-CoV-2 Patient Samples Have Minor Effects on SARS-CoV-2 Detectability by RT-PCR

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for the generation of accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, the impact on SARS-CoV-2 detectability by reverse transcriptase PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2–positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on reverse transcriptase PCR detection. SARS-CoV-2 RNA was still reliably detected by reverse transcriptase PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The change of Ct value per day was 0.023 to 0.046 (±0.018 to 0.019). In addition, viral RNA was still detected after 15 freeze-thaw cycles, with a change of Ct value of 0.106 to 0.197 (±0.009 to 0.061) per freeze-thaw cycle. However, compared with storage at 4°C, RNA was significantly less detectable when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels, as measured by reverse transcriptase PCR assays, are significantly, but not substantially, altered by prolonged unrefrigerated storage of up to 21 days at temperatures ranging up to 35°C or after repeated freeze-thaw cycles.