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Updated: Aug 22, 2025
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Development and validation of RT-PCR assays for testing for SARS-CoV-2
Kanti Pabbaraju1, Anita A Wong1, Mark Douesnard1
1Alberta Precision Laboratories, Public Health Laboratory, Calgary, Alberta, Canada.
Background:
The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays.
Methods:
We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2-specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control.
Results:
These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention.
Conclusions:
The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.
