Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

• Published in: bioRxiv 2021-12
• Main Authors: Craig, Nicky, Fletcher, Sarah Louise, Daniels, Alison, Newman, Caitlin, O'shea, Marie, Warr, Amanda, Tait-Burkard, Christine
• Format: Article
• Language: English
• Subjects: Cell culture; Clinical isolates; High-throughput screening; Lysis; Microbiology; RNA viruses; Severe acute respiratory syndrome coronavirus 2; Viruses
• ISSN: 2692-8205
• DOI: 10.1101/2021.11.30.470550

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro. Competing Interest Statement The authors have declared no competing interest.