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Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
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SARS-CoV-2 RNA Quantification Using Droplet Digital RT-PCR.
Natalie N Kinloch1, Gordon Ritchie2, Winnie Dong3
1Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia; British Columbia Centre for Excellence in HIV/AIDS, Vancouver, British Columbia.
The Journal of Molecular Diagnostics : JMD
|June 1, 2021
Summary
Droplet digital RT-PCR (RT-ddPCR) enables precise SARS-CoV-2 RNA quantification, crucial for COVID-19 control. This method calibrates existing diagnostic data, unlocking deeper insights into SARS-CoV-2 viral load variations.
Area of Science:
- Virology
- Molecular Diagnostics
- Epidemiology
Background:
- Quantitative viral load assays are vital for understanding viral diseases.
- Current SARS-CoV-2 diagnostic tests offer only semiquantitative results.
- Widespread SARS-CoV-2 viral load testing is needed for effective COVID-19 control.
Purpose of the Study:
- To evaluate primer/probe sets for SARS-CoV-2 RNA quantification using RT-ddPCR.
- To establish a method for deriving precise viral load data from existing diagnostic tests.
- To investigate the in vivo variation of SARS-CoV-2 RNA viral loads.
Main Methods:
- Evaluated eight primer/probe sets for RT-ddPCR.
- Determined analytical efficiency, precision, and limits of detection/quantification.
- Calibrated RT-ddPCR results against real-time RT-PCR cycle threshold values.
Main Results:
- Identified three efficient, precise, and sensitive primer/probe sets for RT-ddPCR.
- E-Sarbeco primer/probe set showed ~83% efficiency and ~2% CV at 1000 copies/reaction.
- Observed a 6.2log10 range of SARS-CoV-2 RNA viral loads in patient specimens.
Conclusions:
- RT-ddPCR is a viable platform for accurate SARS-CoV-2 RNA quantification.
- A log-linear relationship allows conversion of RT-PCR data to absolute copy numbers.
- This approach can leverage existing diagnostic data for SARS-CoV-2 research.
