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/.

Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for a pandemic-scale infection surveillance

“…We therefore optimised sample preparation to allow rapid, sensitive, and repeatable testing directly from saliva without purification of RNA (direct RT-LAMP). To avoid time-consuming RNA extractions, we trialled a range of different heat inactivation protocols 21,22 and saliva collection solutions 23,24 , all of which have been found to improve sensitivity in RT-PCR or RT-LAMP assays. However, we adopted a protocol in which fresh or frozen saliva was diluted at least 1 in 4 in AviSal Sample Collection Buffer (Hayat Genetics), followed by virus heat inactivation at 95°C for 10 minutes.…”

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing

“…This is the first report of a high-throughput HI protocol for SARS-CoV-2 testing that is performed on UN733 packaged OP/NP swab samples in bulk. Although the use of heat is an effective means of SARS-CoV-2 inactivation 8 , 11 – 13 , 23 – 25 , COVID-19 testing laboratories have relied on chemical lysis during RNA extraction for viral inactivation. Chemical lysis is highly effective mechanism of virus inactivation 26 , 27 , but requires handling of potentially viable SARS-CoV-2 samples that represents a risk to operators.…”

“…A critical aspect of this work has been an experimental demonstration of virus inactivation using the bulk HI protocol on mock OP/NP swab samples. Insufficient exposure at the required temperature could result in incomplete viral inactivation 13 , 28 which is especially important for samples with high viral load 11 , 12 , 29 . In addition our bulk HI protocol employs wet heat as opposed to dry heat as previously used 30 , which can lead to evaporation and incomplete viral inactivation 28 .…”

“…Such studies have demonstrated that 65 °C for 10 min or 95 °C for 3 min resulted in viral inactivation, however the use of 95 °C for 3 min also resulted in decreased sensitivity in the downstream RT-qPCR assay (2.2 Cq increase) 8 . Others have also observed that at some temperatures, the sensitivity of the RT-qPCR assay is affected, interfering with the diagnosis of patient samples 8 , 16 while in a different study, treatment of 70 °C for up to 30 min did not appear to affect Cq values in nasopharyngeal samples 13 . Therefore a balance between viral inactivation and reduced detection by RT-qPCR must be achieved for any heating protocol.…”

Cotton Fabrics Decorated with Antimicrobial Ag-Coated TiO2 Nanoparticles Are Unable to Fully and Rapidly Eradicate SARS-CoV-2

“…The reverse transcriptase enzyme can then assemble the complementary DNA strand (cDNA) that is amplified subsequently by the DNA polymerase. In this way, Lista et al 30 reported that qRT-PCR amplifies the N1 and N2 genetic sequences of SARS-CoV-2 inactivated or not by heating similarly, even leading to indistinguishable Ct values.…”