Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification

Abstract

Studying the entire virus replication cycle of SARS‐CoV‐2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often re‐quires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and appli‐cable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high‐throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT‐qPCR method for quantification of SARS‐CoV‐2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (‐ve RNA), IAV (segmented ‐ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped vi‐ruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS‐CoV‐2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high‐ and low‐throughput applications.

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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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DOI

https://doi.org/10.3390/v14030508

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https://www.research.ed.ac.uk/en/publications/5778a254-841d-4f3e-a278-f81edf73a416