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Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.
- Source :
-
PloS one [PLoS One] 2021 Sep 15; Vol. 16 (9), pp. e0256813. Date of Electronic Publication: 2021 Sep 15 (Print Publication: 2021). - Publication Year :
- 2021
-
Abstract
- There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.<br />Competing Interests: The authors have declared that no competing interests exist.
- Subjects :
- COVID-19 epidemiology
COVID-19 virology
Epidemics prevention & control
Humans
Nasopharynx virology
Reagent Kits, Diagnostic
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction methods
SARS-CoV-2 physiology
Sensitivity and Specificity
Specimen Handling methods
Workflow
COVID-19 diagnosis
COVID-19 Testing methods
Hot Temperature
RNA, Viral genetics
SARS-CoV-2 genetics
Virus Inactivation
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 16
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 34525109
- Full Text :
- https://doi.org/10.1371/journal.pone.0256813