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

Abstract

There is a worldwide shortage of reagents to perform detection of SARS-CoV-2. Many clinical diagnostic laboratories rely on commercial platforms that provide integrated end-to-end solutions. While this provides established robust pipelines, there is a clear bottleneck in the supply of reagents given the current situation of extraordinary high demand. Some laboratories resort to implementing kit-free handling procedures, but many other small laboratories will not have the capacity to develop those and/or will perform manual handling of their samples. In order to provide multiple workflows for SARS-CoV-2 nucleic acid detection we compared several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAGEN), the recently developed RNAdvance Blood (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared different 1-step RT-qPCR Master Mix brands: TaqMan™ Fast Virus 1-Step Master Mix (ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (NEB). We used the Centre for Disease Control (CDC) recommended primers that detect two regions of the viral N gene as well as those that detect the RdRP gene region as per Public Health England (PHE) guidelines (Charité/WHO/PHE). Our data show that the RNA extraction methods provide similar results. Amongst the qPCR reagents tested, TaqMan™ Fast Virus 1-Step Master Mix and Luna® Universal Probe One-Step RT-qPCR Kit proved most sensitive. The N1 and N2 primer-probes provide a more reliable detection than the RdRP_SARSr primer-probe set, particularly in samples with low viral titres. Importantly, we have implemented a protocol using heat inactivation and demonstrate that it has minimal impact on the sensitivity of the qPCR in clinical samples -potentially making SARS-CoV-2 testing portable to 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/.

Estimates of the rate of infection and asymptomatic COVID-19 disease in a population sample from SE England

“…Buccal swabs obtained during the home visits were used to test for current SARS-CoV-2 RNA (and by inference active replication) using RT-PCR as previously described by Lista et al 10 Briefly, nasopharyngeal swab samples were heat inactivated at 70 °C for 30 min and 140 μl were used to extract RNA with the Beckman Coulter RNAdvance Blood kit, ending with elution in 50 μL of water. For qPCR, the US CDC designed primer/probe set were used for the N gene (N1 and N2) and RNAseP with 5 μL of RNA sample and Taqman Fast Virus 1-Step Master Mix (Thermofisher).…”

‘All In’: a pragmatic framework for COVID‐19 testing and action on a global scale

“…If sufficiently characterized for screening, this and other newly developing approaches could allow for more rapid and less reagent intensive virologic analysis of swabbed samples than the standard RT–PCR testing approaches that have been broadly employed for COVID testing (e.g., the majority of in vitro diagnostic tests receiving an Emergency Use Authorization (EUA) approval from the US FDA and/or are recommended by health authorities in other countries rely on RT–PCR methods with an RNA extraction step). Some of these labs are also exploring means to partially or totally inactivate the virus just after swab collection in order to increase the throughput of these more rapid RT – PCR methods and expand the number of labs eligible to process samples (preprint: Lista et al , ). Optimization of sampling procedures via protocols for alternative swabs and sampling media may also contribute to the propagation of testing via rapid RT – PCR or other means.…”

Evaluation of Commercially Available Viral Transport Medium (VTM) for SARS-CoV-2 Inactivation and Use in Point-of-Care (POC) Testing

“…COPAN™, another VTM, is registered as a non-propagating transport and culture medium with the FDA but does not contain any inactivating agents [ 11 ]. To date, there have been nine studies that have evaluated chemical inactivating ingredients and shown effective virucidal activity against SARS-CoV-2 [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ], but to our knowledge there has been no evaluation of commercially available VTMs that could be utilized in an appropriate workflow for community-based molecular POC testing.…”