M. Lista, Robert Page, Helin Sertkaya, P. Matos, E. Ortiz-Zapater, T. Maguire, Kate Poulton, Aoife O'Byrne, Clément R. Bouton, Ruth E Dickenson, M. Ficarelli, M. Howard, G. Betancor, R. Galão, S. Pickering, A. Signell, H. Wilson, P. Cliff, Amita Patel, E. Macmahon, E. Cunningham, M. Agromayor, J. Martin-Serrano, E. Perucha, Hannah E Mischo, M. Shankar-Hari, R. Batra, J. Edgeworth, M. Malim, S. Neil, R. Martinez-Nunez
medRxiv, 2020
There is a worldwide shortage of reagents to perform detection of SARS-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 (Charite/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.
Cited by 16 publications.
Field of study: Medicine