Journal:Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples
Full article title | Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples |
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Journal | International Journal of Infectious Diseases |
Author(s) |
Alcoba-Florez, Julia; González-Montelongo, Rafaela; Íñigo-Campos, A.; García-Martínezde Artola, Diego; Gil-Campesino, Helena; The Microbiology Technical Support Team; Ciuffreda, Laura; Valenzuela-Fernández, Agustín; Flores, Carlos |
Author affiliation(s) |
Hospital Universitario Nuestra Señora de Candelaria, Instituto Tecnológico y de Energías Renovables, Universidad de La Laguna, Instituto de Salud Carlos III |
Primary contact | Email: cflores at ull dot edu dot es |
Year published | 2020 |
Volume and issue | 97 |
Page(s) | 66–68 |
DOI | 10.3389/fcell.2020.00468 |
ISSN | 2296-634X |
Distribution license | Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International |
Website | https://www.sciencedirect.com/science/article/pii/S1201971220304069 |
Download | https://www.sciencedirect.com/science/article/pii/S1201971220304069/pdfft (PDF) |
This article should be considered a work in progress and incomplete. Consider this article incomplete until this notice is removed. |
Abstract
Objectives: The gold-standard COVID-19 diagnosis relies on detecting SARS-CoV-2 using RNA purification and one-step retrotranscription and quantitative real-time PCR (RT-qPCR). Based on the urgent need for high-throughput screening, we tested the performance of three alternative protocols that are simple and affordable for rapidly detecting SARS-CoV-2, bypassing the long and tedious RNA extraction step and reducing the time to viral detection.
Methods: We evaluated three methods based on direct nasopharyngeal swab viral transmission medium (VTM) heating before RT-qPCR: a) direct without additives; b) in a formamide-EDTA (FAE) buffer, and c) in a RNAsnap buffer.
Results: Although with a delay in cycle threshold compared to the gold-standard, we found consistent results in nasopharyngeal swab samples that were subject to a direct 70°C incubation for 10 minutes.
Conclusions: Our findings provide valuable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests, thereby complementing standard diagnosis.
Keywords: COVID-19, SARS-CoV-2, diagnosis, sample treatment, RNA extraction, fast protocols
Introduction
The ongoing coronavirus disease 2019 (COVID-19) worldwide pandemic being caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus (see WHO situation reports for more) has imposed an unexpected high burden on health care systems around the world, leading to an increasing demand for daily diagnostic screening. The current standard assay for diagnosis is based on the extraction of RNA from respiratory samples—especially from nasopharyngeal swab viral transport media (VTM)—and subsequent one-step reverse transcription and real-time quantitative PCR (RT-qPCR) targeting one or several sequences from SARS-CoV-2. (Corman et al. 2020) However, this standard procedure usually takes from 3.5 to 4.0 hours due to manual interventions. Additionally, there is a risk of reagent shortage in major kit suppliers, particularly for the RNA extraction step. Alternatives to accelerate this procedure have been proposed as a consequence, with the most efficient alternative relying on loop-mediated isothermal amplification (LAMP). (Esbin et al. 2020)
References
Notes
This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added. References in this version are listed in order of appearance—by design—rather than alphabetical order as the original was.