Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification
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 requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinic...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-02-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/14/3/508 |
| _version_ | 1797441006116798464 |
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| author | Nicky Craig Sarah L. Fletcher Alison Daniels Caitlin Newman Marie O’Shea Wenfang Spring Tan Amanda Warr Christine Tait-Burkard |
| author_facet | Nicky Craig Sarah L. Fletcher Alison Daniels Caitlin Newman Marie O’Shea Wenfang Spring Tan Amanda Warr Christine Tait-Burkard |
| author_sort | Nicky Craig |
| collection | DOAJ |
| description | 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 requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable 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 TCID<sub>50</sub> 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 viruses, 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. |
| first_indexed | 2024-03-09T12:16:40Z |
| format | Article |
| id | doaj.art-464657a544674df28b89261194a445a8 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-09T12:16:40Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-464657a544674df28b89261194a445a82023-11-30T22:45:34ZengMDPI AGViruses1999-49152022-02-0114350810.3390/v14030508Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate QuantificationNicky Craig0Sarah L. Fletcher1Alison Daniels2Caitlin Newman3Marie O’Shea4Wenfang Spring Tan5Amanda Warr6Christine Tait-Burkard7The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKThe Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UKStudying 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 requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable 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 TCID<sub>50</sub> 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 viruses, 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.https://www.mdpi.com/1999-4915/14/3/508SARS-CoV-2real-time PCRCOVID-19direct lysisdiagnosticscoronavirus |
| spellingShingle | Nicky Craig Sarah L. Fletcher Alison Daniels Caitlin Newman Marie O’Shea Wenfang Spring Tan Amanda Warr Christine Tait-Burkard Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification Viruses SARS-CoV-2 real-time PCR COVID-19 direct lysis diagnostics coronavirus |
| title | Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification |
| title_full | Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification |
| title_fullStr | Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification |
| title_full_unstemmed | Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification |
| title_short | Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification |
| title_sort | direct lysis rt qpcr of sars cov 2 in cell culture supernatant allows for fast and accurate quantification |
| topic | SARS-CoV-2 real-time PCR COVID-19 direct lysis diagnostics coronavirus |
| url | https://www.mdpi.com/1999-4915/14/3/508 |
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