Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR

• Published in: Nature communications Vol. 11; no. 1; pp. 4812 - 12
• Main Authors: Smyrlaki, Ioanna, Ekman, Martin, Lentini, Antonio, Rufino de Sousa, Nuno, Papanicolaou, Natali, Vondracek, Martin, Aarum, Johan, Safari, Hamzah, Muradrasoli, Shaman, Rothfuchs, Antonio Gigliotti, Albert, Jan, Högberg, Björn, Reinius, Björn
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.09.2020; Nature Portfolio
• Subjects: 38/77; 38/88; 631/326/596/4130; 692/699/255/2514; 692/700/139/1420; Benchmarking; Betacoronavirus - genetics; Betacoronavirus - isolation & purification; Clinical Laboratory Techniques - methods; Clinical Laboratory Techniques - standards; Clinical Laboratory Techniques - statistics & numerical data; Coronavirus Infections - diagnosis; Coronavirus Infections - epidemiology; Coronavirus Infections - virology; COVID-19; COVID-19 Testing; DNA Primers - genetics; Hot Temperature; Humanities and Social Sciences; Humans; multidisciplinary; Pandemics; Pneumonia, Viral - diagnosis; Pneumonia, Viral - epidemiology; Pneumonia, Viral - virology; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; Reverse Transcriptase Polymerase Chain Reaction - statistics & numerical data; RNA, Viral - genetics; RNA, Viral - isolation & purification; SARS-CoV-2; Science; Science (multidisciplinary); Sensitivity and Specificity; Sweden - epidemiology; Viral Plaque Assay - methods; Sweden
• DOI: 10.1038/s41467-020-18611-5
• ISSN: 2041-1723

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing. SARS-CoV-2 infection is widely diagnosed by RT-PCR, but RNA extraction is a bottleneck for fast and cheap diagnosis. Here, the authors develop protocols to perform RT-PCR directly on heat-inactivated subject samples or samples lysed with readily available detergents and benchmark performance against 597 clinically diagnosed patient samples.