About: We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.   Goto Sponge  NotDistinct  Permalink

An Entity of Type : fabio:Abstract, within Data Space : wasabi.inria.fr associated with source document(s)

AttributesValues
type
value
  • We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×106 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.
subject
  • Virology
  • Medical research institutes in the United States
  • Molecular biology
  • Forensic genetics
part of
is abstract of
is hasSource of
Faceted Search & Find service v1.13.91 as of Mar 24 2020


Alternative Linked Data Documents: Sponger | ODE     Content Formats:       RDF       ODATA       Microdata      About   
This material is Open Knowledge   W3C Semantic Web Technology [RDF Data]
OpenLink Virtuoso version 07.20.3229 as of Jul 10 2020, on Linux (x86_64-pc-linux-gnu), Single-Server Edition (94 GB total memory)
Data on this page belongs to its respective rights holders.
Virtuoso Faceted Browser Copyright © 2009-2025 OpenLink Software