In:
PLOS ONE, Public Library of Science (PLoS), Vol. 16, No. 9 ( 2021-9-15), p. e0256813-
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/ .
Type of Medium:
Online Resource
ISSN:
1932-6203
DOI:
10.1371/journal.pone.0256813
DOI:
10.1371/journal.pone.0256813.g001
DOI:
10.1371/journal.pone.0256813.g002
DOI:
10.1371/journal.pone.0256813.g003
DOI:
10.1371/journal.pone.0256813.g004
DOI:
10.1371/journal.pone.0256813.g005
DOI:
10.1371/journal.pone.0256813.s001
DOI:
10.1371/journal.pone.0256813.s002
DOI:
10.1371/journal.pone.0256813.s003
DOI:
10.1371/journal.pone.0256813.r001
DOI:
10.1371/journal.pone.0256813.r002
DOI:
10.1371/journal.pone.0256813.r003
DOI:
10.1371/journal.pone.0256813.r004
DOI:
10.1371/journal.pone.0256813.r005
Language:
English
Publisher:
Public Library of Science (PLoS)
Publication Date:
2021
detail.hit.zdb_id:
2267670-3
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