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.
【저자키워드】 SARS-CoV-2, viral infection, Laboratory techniques and procedures, 【초록키워드】 COVID-19, infection control, Coronavirus disease 2019, coronavirus, protocol, SARS-COV-2 infection, diagnostic, Diagnosis, RT-PCR, RNA extraction, COVID-19 testing, heat inactivation, Patient, Effectiveness, reverse transcription, Viral RNA, Plaque assay, Efficiency, Transport, Patient care, acute respiratory syndrome, subject, medium, PCR-based testing, buffer, heat-inactivated, polymerase chain, detect, develop, evaluate, caused, clinically, diagnosed, lysed, Significant, 【제목키워드】 COVID-19 testing, SARS-CoV-2 RT-PCR, massive, feasible,