tetano
Editor, Senior Moderator
J Virol Methods
. 2021 Aug 8;114251.
doi: 10.1016/j.jviromet.2021.114251. Online ahead of print.
Development of a Rapid Viability RT-PCR (RV-RT-PCR) Method to Detect Infectious SARS-CoV-2 from Swabs
Sanjiv R Shah[SUP] 1 [/SUP], Staci R Kane[SUP] 2 [/SUP], Maher Elsheikh[SUP] 3 [/SUP], Teneile M Alfaro[SUP] 3 [/SUP]
Affiliations
Abstract
Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (C[SUB]T[/SUB]) difference (ΔC[SUB]T[/SUB]). An algorithm based on ΔC[SUB]T[/SUB] ≥ 6 representing ∼ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 hours (for a batch of 12 swabs), compared to days typically required by the cell-culture based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.
Keywords: COVID-19; Reverse Transcriptase PCR; infectious SARS-CoV-2; rapid viability; viral detection.
. 2021 Aug 8;114251.
doi: 10.1016/j.jviromet.2021.114251. Online ahead of print.
Development of a Rapid Viability RT-PCR (RV-RT-PCR) Method to Detect Infectious SARS-CoV-2 from Swabs
Sanjiv R Shah[SUP] 1 [/SUP], Staci R Kane[SUP] 2 [/SUP], Maher Elsheikh[SUP] 3 [/SUP], Teneile M Alfaro[SUP] 3 [/SUP]
Affiliations
- PMID: 34380012
- DOI: 10.1016/j.jviromet.2021.114251
Abstract
Since the rapid onset of the COVID-19 pandemic, its causative virus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), continues to spread and increase the number of fatalities. To expedite studies on understanding potential surface transmission of the virus and to aid environmental epidemiological investigations, we developed a rapid viability reverse transcriptase PCR (RV-RT-PCR) method that detects viable (infectious) SARS-CoV-2 from swab samples in <1 day compared to several days required by current gold-standard cell-culture-based methods. The method integrates cell-culture-based viral enrichment in a 96-well plate format with gene-specific RT-PCR-based analysis before and after sample incubation to determine the cycle threshold (C[SUB]T[/SUB]) difference (ΔC[SUB]T[/SUB]). An algorithm based on ΔC[SUB]T[/SUB] ≥ 6 representing ∼ 2-log or more increase in SARS-CoV-2 RNA following enrichment determines the presence of infectious virus. The RV-RT-PCR method with 2-hr viral infection and 9-hr post-infection incubation periods includes ultrafiltration to concentrate virions, resulting in detection of <50 SARS-CoV-2 virions in swab samples in 17 hours (for a batch of 12 swabs), compared to days typically required by the cell-culture based method. The SARS-CoV-2 RV-RT-PCR method may also be useful in clinical sample analysis and antiviral drug testing, and could serve as a model for developing rapid methods for other viruses of concern.
Keywords: COVID-19; Reverse Transcriptase PCR; infectious SARS-CoV-2; rapid viability; viral detection.