Prolonged Unfrozen Storage and Repeated Freeze-Thawing of SARS-CoV-2 Patient Samples Have Minor Effects on SARS-CoV-2 Detectability by RT-PCR.
Author(s): Dzung A, Cheng P, Stoffel C, Tastanova A, Turko P, Levesque MP, Bosshard PP
Publication: J Mol Diagn, 2021, Vol. , Page
PubMed ID: 33775845 PubMed Review Paper? No
Purpose of Paper
This paper compared cycle threshold (CT) values for SARS-CoV-2, the virus that causes the novel 2019 coronavirus disease (COVID, COVID-19, COVID19), in respiratory specimens over the course of storage for up to 21 days at three temperatures and after up to 15 freeze/thaw cycles.
Conclusion of Paper
SARS-CoV-2 RNA was detected in all specimens, regardless of storage temperature or duration, but small changes in CT values (0.002-0.046 cycles per day) were noted. The change in CT value for each of the three genes was smaller when stored at 4°C than 35°C or 25°C. Larger changes in CT values were observed when specimens were freeze/thaw cycled five times compared to those only freeze/thaw cycled once and CT values increased further when subjected to ten freeze/thaw cycles.
Studies
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Study Purpose
This study compared CT values for three SARS-CoV-2 genes in respiratory specimens over the course of storage for up to 21 days at three temperatures and after up to 15 freeze-thaw cycles. The study included residual nasopharyngeal swab specimens collected into viral transport media (VTM) from five patients with SARS-CoV-2. Specimens were stored for <30 days at -20°C before they were thawed, diluted, and aliquoted into cryotubes for this study. Aliquots were stored at 4, 25, or 35°C for 1, 2, 3, 4, 5, 6, 7, 10, 14, or 21 days and then stored at -80°C or were frozen in liquid nitrogen and thawed at room temperature 1, 5, 10, or 15 times before storage. RNA was extracted from all specimens using the KingFisher Flex Purification System. SARS-CoV-2 ORF1ab, S gene, and N genes were detected using the real-time PCR TaqMan SARS-CoV-2 Assay Kit v2.
Summary of Findings:
SARS-CoV-2 RNA was detected in all specimens, regardless of storage temperature or duration, but small changes in CT values (0.002-0.046 cycles per day) were noted. The change in CT value for each of the three genes was smaller when stored at 4°C (0.002-0.006 cycles/day) than 35°C (0.023-0.046 cycles/day, P<0.0001) or 25°C (0.008-0.036 cycles/day, P<0.0001). CT values were significantly higher in specimens subjected to five freeze/thaw cycles compared to those only freeze/thaw cycled once (P=0.0005). Specimens subjected to ten freeze/thaw cycles had higher CT values than those subjected to five cycles (P<0.0001) but CT values were comparable in specimens subjected to ten and 15 freeze/thaw cycles. The authors found for every freeze/thaw cycle, the average CT value of the N-gene, ORF1ab, and S genes increased by 0.197, 0.127, and 0.106 cycles, respectively.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Pneumonia/Respiratory Infection
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 days
1 day
2 days
3 days
4 days
5 days
6 days
7 days
10 days
14 days
21 days
Storage Freeze/thaw cycling 1 cycle
5 cycles
10 cycles
15 cycles
Real-time qRT-PCR Specific Targeted nucleic acid ORF1ab
S gene
N gene
Storage Storage temperature 25°C
35°C