The World Health Organization has declared COVID-19 caused by the newly discovered SARS-CoV-2 a pandemic. Due to growing demand for reagents and/ or kits to extract SARS-CoV-2 RNA for subsequent RT-qPCR diagnostics, there is a worldwide risk of shortages. With a detection sensitivity of 97.4% (95% CI: 86.2-99.9%), we describe a simple, fast, alternative workflow for molecular detection of SARS-CoV-2, where samples are simply heat-processed for 5 min at 98 °C before a commonly-used RT-qPCR procedure.Coronavirus disease (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), was first detected in Wuhan, China in December 2019 and then spread worldwide in a few months [1]. There is currently a global shortage of viral nucleic acid (NA) extraction kits, which is affecting the diagnosis of an increasing number of suspected COVID-19 cases. The aim of this study was to investigate a new simplified workflow for molecular detection of SARS-CoV-2 that does not require NA extraction and could serve as an alternative in diagnostic laboratories to overcome chemical-based kit-shortages.
Direct approach for molecular detection of SARS-CoV-2NA purification before PCR/reverse transcription (RT)-PCR is the gold standard for molecular diagnostics. The MagNa Pure 96 system (Roche Molecular Biochemicals, Indianapolis, Indiana, United States (US)) is a widely used system for high-throughput NA purification in many public health laboratories worldwide [2]. However, with Roche's announcement of emerging kitshortages and bottlenecks in kit production processes [3], we investigated if real-time RT-PCR (RT-qPCR) analysis could be performed with minimal pre-treatment of samples. We used the most common sample type (oropharyngeal swabs) collected from patients suspected of COVID-19 in Denmark.Three simplified approaches, which involved minimal handling of the samples before the RT-qPCR for SARS-CoV-2 were employed to avoid the NA purification step. The first approach was direct: 5 µL of the saline/transport solution from the throat-swab were added to the RT-qPCR reaction without any treatment. The second was a phosphate-buffered saline (PBS) diluted approach: the saline/transport solution was further diluted 1:1 with PBS before adding 5 µL directly to the RT-qPCR reaction. The third was a heat-processed method: we compared four different heat-processes on 10 µL of the saline/transport solution from the throat swab, (i) 5 min at 95 °C, (ii) 10 min at 95 °C, (iii) 5 min at 98 °C and (iv) 10 min at 98 °C, respectively. All heat-processed clinical samples were cooled for 2 min at 4 °C before 5 µL were used in the RT-qPCR reaction. Two SARS-CoV-2 RT-qPCR assays were used: (i) the published and widely used RT-qPCR assay for the envelope (E)-gene [4,5] combined with the SensiFAST Probe No-ROX One-Step Real-time PCR kit (Bioline Meridian BioScience, Cincinnati, Ohio, US), and (ii) the commercial RealStar SARS-CoV-2 RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany). We employed 87 patient samples, comprising 6...