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Updated: Nov 8, 2025
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Raphael Nyaruaba1, Xiaohong Li2, Caroline Mwaliko3
1Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences; International College, University of Chinese Academy of Sciences; Sino-Africa Joint Research Center; rohuru1@gmail.com.
Droplet digital PCR (ddPCR) offers superior SARS-CoV-2 detection compared to RT-qPCR, especially for low-abundance targets. This study details developing multiplex ddPCR assays for sensitive, high-throughput viral diagnosis.
Area of Science:
- Molecular Biology
- Infectious Disease Diagnostics
- Genomics
Background:
- The SARS-CoV-2 pandemic necessitates accurate and sensitive diagnostic methods.
- Reverse transcription quantitative PCR (RT-qPCR) is the current standard but has limitations with low-abundance targets.
- Droplet digital PCR (ddPCR) presents an emerging technology with enhanced sensitivity for nucleic acid quantification.
Purpose of the Study:
- To expand the capabilities of RT-ddPCR for SARS-CoV-2 diagnosis.
- To demonstrate the development of simplex, duplex, triplex, and quadruplex ddPCR assays.
- To provide detailed protocols for sensitive detection of multiple SARS-CoV-2 targets.
Main Methods:
- Development of multiplex probe mixes targeting specific SARS-CoV-2 genome regions (N, ORF1ab, RPP30, RBD2).
- Utilized a two-color detection system for multiplexing ddPCR assays.
- Provided step-by-step protocols, notes, and data analysis suggestions for assay optimization.
Main Results:
- Successfully developed simplex to quadruplex ddPCR assays for SARS-CoV-2 detection.
- Demonstrated the feasibility of using primers and probes targeting multiple viral sites.
- Established a workflow for sensitive detection of multiple targets in small samples.
Conclusions:
- RT-ddPCR offers significant advantages over RT-qPCR for SARS-CoV-2 diagnosis, particularly with low viral loads.
- The developed multiplex assay workflow enhances sensitivity, cost-effectiveness, and sample throughput.
- This adaptable workflow can improve the detection of multiple targets in future diagnostic applications.
