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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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Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing.
Raphael Nyaruaba1,2,3, Changchang Li1,2, Caroline Mwaliko1,2,3
1Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, China.
Expert Review of Molecular Diagnostics
|December 31, 2020
Summary
Multiplex droplet digital PCR (ddPCR) assays were developed for SARS-CoV-2 detection and antiviral screening. These multiplex ddPCR assays demonstrate superior sensitivity and enable efficient drug efficacy testing.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Droplet digital PCR (ddPCR) offers superior sensitivity for SARS-CoV-2 detection compared to traditional RT-PCR.
- Limited research exists on developing and evaluating multiplex ddPCR assays for SARS-CoV-2.
- Multiplex assays can enhance efficiency in diagnostics and antiviral screening.
Purpose of the Study:
- To develop and optimize simplex, duplex, triplex, and quadruplex ddPCR assays for SARS-CoV-2 detection.
- To evaluate the performance of these multiplex ddPCR assays, including their sensitivity and accuracy.
- To assess the utility of multiplex ddPCR for antiviral screening.
Main Methods:
- Development and optimization of singleplex, duplex, triplex, and quadruplex ddPCR assays using multiple primer/probe sets.
- Utilized a two-color ddPCR detection system for assay development.
- Performance analysis included limit of detection, accuracy, and testing on 94 clinical samples.
Main Results:
- The quadruplex ddPCR assay demonstrated comparable limits of detection and accuracy to lower-order multiplex assays.
- The ddPCR triplex probe mix assay exhibited higher sensitivity than RT-qPCR when analyzing clinical samples.
- In vitro antiviral screening identified remdesivir as an inhibitor of SARS-CoV-2, while another drug did not show inhibition.
Conclusions:
- Multiplex ddPCR assays for SARS-CoV-2 detection and antiviral screening are feasible through probe mix and amplitude-based multiplexing.
- Developed multiplex ddPCR assays offer enhanced sensitivity and efficiency for various SARS-CoV-2 applications.
- This approach facilitates advanced applications such as high-throughput antiviral drug screening.
