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Updated: Oct 2, 2025
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Retrospective quantitative detection of SARS-CoV-2 by digital PCR showing high accuracy for low viral load specimens
Renfei Lu1, Jian Wang1, Min Li1
1Clinical Laboratory, the Nantong Third Hospital Affiliated to Nantong University, Nantong, P. R. China.
Introduction:
Accurate detection of severe acute respiratory syndrome coronavirus 2 is critical for diagnosis and disease status evaluation of Coronavirus disease 2019. We retrospectively evaluated the infection status and viral load of severe acute respiratory syndrome coronavirus 2 in Nantong city, China, using a quantitative digital polymerase chain reaction and reverse-transcription PCR.
Methodology:
A total of 103 clinical specimens from 31 patients were collected and tested by digital PCR and reverse-transcription PCR.
Results:
The overall accuracy of digital PCR was 96.8%, which was higher than the overall accuracy of 87.1% for reverse-transcription PCR. 4 (3.88%) specimens for ORF1ab and 22 (21.36%) specimens for N gene were negative by reverse-transcription PCR but positive by digital PCR. 3 (2.91%, 3/103) specimens of ORF1ab were positive by reverse-transcription PCR but negative by digital PCR. The digital PCR assay exhibited higher sensitivity to measure the N gene than the ORF1ab gene (p < 0.01).
Conclusions:
Our results showed that digital PCR assay provides more reliable detection of Coronavirus disease 2019 than reverse-transcription PCR, especially for low viral load specimens.
