Highlights High-throughput screening of SARS-CoV-2 by RT-qPCR without previous RNA extraction. Heated nasopharyngeal swab transport medium as template in the RT-qPCR test. Fast and safety management of SARS-CoV-2 samples in the diagnostic process.

The current reference for COVID-19 diagnosis is based on the detection of SARS-CoV-2 on RNA extracts using one-step retrotranscription and quantitative PCR (RT-qPCR). Based on the urgent need for high-throughput COVID-19 screening, we tested the performance of three alternative, simple and affordable protocols to rapidly detect SARS-CoV-2, overcoming the long and tedious RNA extraction step. Although with an average increase of 6.1 (± 1.6) cycles compared to standard tests with RNA extracts, we show that RT-qPCR yielded consistent results in nasopharyngeal swab samples that were subject to a direct 70 o C incubation for 10 min. Our findings provide viable options to overcome any supply chain issue and help to increase the throughput of diagnostic tests by using any qPCR device, thereby complementing standard COVID-19 testing.

Highlights This study compared RT-qPCR sensitivity for SARS-CoV-2 detection. False negatives were in the range of 2–39.8%. The most sensitive solution (97.9% [92.8–99.7]) targeted the SARS-CoV-2 E-gene.

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Objectives: The ongoing COVID-19 pandemic continues imposing a demand for diagnostic screening. In anticipation that the recurrence of outbreaks and the measures for lifting the lockdown worldwide may cause supply chain issues over the coming months, we assessed the sensitivity of a number of one-step retrotranscription and quantitative PCR (RT-qPCR) solutions to detect SARS-CoV-2. Methods: We evaluated six different RT-qPCR alternatives for SARS-CoV-2/COVID-19 diagnosis based on standard RNA extractions. That of best sensitivity was also assessed with direct nasopharyngeal swab viral transmission medium (VTM) heating, overcoming the RNA extraction step. Results: We found a wide variability in the sensitivity of RT-qPCR solutions that associated with a range of false negatives from as low as 2% (0.3-7.9%) to as much as 39.8% (30.2-50.2). Direct preheating of VTM combined with the best solution provided a sensitivity of 72.5% (62.5-81.0), in the range of some of the solutions based on standard RNA extractions. Conclusions: We evidenced sensitivity limitations of currently used RT-qPCR solutions. Our results will help to calibrate the impact of false negative diagnoses of COVID-19, and to detect and control new SARS-CoV-2 outbreaks and community transmissions.