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Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

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/.

SUBMITTER: Lista MJ

PROVIDER: S-EPMC8443028 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples.MethodsA virus panel based on the principle of biotinylated RNA baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2-positive samples. Libraries generated from complex samples were sequenced via generic HTS (without enrichment) and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted.ResultsThe VirBaits set consists of 177,471 RNA baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also points to the risk of cross-contamination between samples.ConclusionsThe VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector, or research question. The risk of cross-contamination needs to be taken into consideration. The application of the RNA-baits principle turned out to be user friendly, and even non-experts can easily use the VirBaits workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible as shown for SARS-CoV-2. Video abstract.

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Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

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