SARS-CoV-2 detection by direct rRT-PCR without RNA extraction.
Author(s): Merindol N, Pépin G, Marchand C, Rheault M, Peterson C, Poirier A, Houle C, Germain H, Danylo A
Publication: J Clin Virol, 2020, Vol. 128, Page 104423
PubMed ID: 32416598 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of collection media, amplification without RNA extraction, and assay used on the detection of SARS-CoV-2, the virus that causes the novel 2019 coronavirus disease (COVID, COVID-19, COVID19). The effects of post-thaw storage and dilution of RNA in water or saline were also investigated.
Conclusion of Paper
Generally, SARS-CoV-2 detection was consistent between the two real-time RT PCR kits with concordant results obtained for 87 of the 88 specimens using the Abbott Kit and SeeGene Kit. Although SARS-CoV-2, was detected using the SeeGene assay in all specimens stored in universal transport media (UTM) at -80°C, regardless of extraction, SARS-CoV-2 was detected using SeeGene in only 65 of the 95 specimens following collection in Hank’s media when extraction wasn’t performed. Similarly, amplification was possible in 30-32 and 24-27 of the 39 specimens stored in water using the SeeGene and Altona kits, respectively, with slightly lower success for the S-gene observed using the Altona Kit when no RNA extraction was performed. Importantly, the specimens stored in water for which SARS-CoV-2 could not be detected after thawing had a higher average initial cycle threshold (CT) values than those with detection after thawing. Average CT values were slightly higher for specimens stored in water or Hank’s media but not UTM when extraction was not performed. Specimens in UTM frozen at -80°C overnight and then thawed and stored at 4°C for up to 72 h had similar CT values for all three genes in the SeeGene assay as specimens that were never frozen. Dilution of extracted RNA in saline water rather than water resulted in >10 increase in CT values for the N and RDRP genes and loss of detection of the E gene.
Studies
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Study Purpose
This study investigated the effects of collection media, amplification without RNA extraction, and assay used on the detection of SARS-CoV-2. The effects of post-thaw storage and dilution of RNA in water or saline were also investigated. Oro- and naso-pharyngeal swabs from an unspecified number of patients with symptoms consistent with SARS-CoV-2 infection were placed in UTM (≥22 SARS CoV-2 positive specimens), Hank’s media (95 SARS-CoV-2 positive specimens), or water (≥ 39 SARS-CoV-2 positive specimens). Virus was inactivated by heating to 95°C for 5 min, stored at 4°C, and unless otherwise specified, were then transferred to -80°C. RNA was extracted using the Abbott mSample Preparation Systems DNA Kit on m2000sp instrument and stored at -80°C. Virus was detected in extracted RNA and directly from media using the m2000rt using the SeeGene Kit and Altona Kit. Positive was defined as CT<40 for any viral gene. To determine the effects of post-thaw storage, some specimens in UTM were frozen overnight at -80°C, thawed, and stored at 4°C for 0, 24, 48, or 72 h before real-time RT-PCR analysis (no extraction). To determine the effects of diluting, RNA purified from five SARS-CoV-2 positive specimens was diluted 1:100 in molecular water and saline.
Summary of Findings:
SARS-CoV-2 detection was consistent between the two real-time RT PCR kits with concordant results obtained for 87 of the 88 specimens. The discordant specimen was positive using the Abbot Kit (CT 35.6 for S gene only) but negative using SeeGene. SARS-CoV-2 was detected with or without extraction in all 17 swabs collected in UTM and stored at -80°C using the SeeGene assay but while the N-gene was amplified in all 17 specimens regardless of extraction, the E-gene and RDRP genes were only detected in 14 specimens without extraction. SARS-CoV-2 was detected using SeeGene in all 30 specimens collected in Hank’s media when RNA extraction was performed but only in 65 of the 95 SARS-CoV-2 positive specimens when no extraction was performed. An initial study found swabs collected in water had comparable detection of the N, E, and RDRP genes using the SeeGene Kit with and without extraction. Further investigation of 39 SARS-CoV-2 positive specimens found amplification using the SeeGene Kit in 30-32 of the specimens stored in water, regardless of whether extraction was performed, but amplification of the E and S gene using the Altona Kit was successful in 24 and 27 specimens stored in water, respectively, and in 26 and 27 RNA extracts from the same specimens, respectively. The specimens stored in water for which SARS-CoV-2 could not be detected after thawing had a higher average initial CT value than those with detection after thawing (25.69 vs 33.7, P< 0.0001 using Altona and 25.97 vs 35.68, P< 0.0001 using SeeGene). Specimens in UTM frozen at -80°C overnight and then thawed and stored at 4°C for up to 72 h had similar CT values for all three genes in the SeeGene assay as specimens that were never frozen. Dilution of extracted RNA in saline water rather than water resulted in >10 increase in CT values for the N and RDRP genes and loss of detection of the E gene.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Pneumonia/Respiratory Infection
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution UTM
Hank's media
Water
Biospecimen Preservation Type of fixation/preservation Frozen
Refrigeration
Analyte Extraction and Purification Analyte isolation method RNA extracted
No extraction performed
Storage Thaw duration 0 h
24 h
48 h
72 h
Real-time qRT-PCR Specific Technology platform SeeGene assay
Altona assay
Real-time qRT-PCR Specific Targeted nucleic acid SARS-CoV-2 E and S genes (Altona)
SARS-CoV-2 E, N and RDRP genes (SeeGene)
Real-time qRT-PCR Specific Template modification Diluted in saline
Diluted in water