TY - JOUR
AU - Craig, Nicky
AU - Fletcher, Sarah
AU - Daniels, Alison
AU - Newman, Caitlin
AU - O'Shea, Marie
AU - Tan, Wenfang Spring
AU - Warr, Amanda
AU - Tait-Burkard, Christine
PY - 2022
DA - February
TI - Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification
JO - Viruses
VL - 14
IS - 3
DO - https://doi.org/10.3390/v14030508
AB - 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 &lt;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.
PB - MDPI
UR - https://www.research.ed.ac.uk/en/publications/5778a254-841d-4f3e-a278-f81edf73a416
KW - Coronavirus (COVID-19)
ER