Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

Keywords: COVID-19; RT-dPCR; RT-qPCR; SARS-CoV-2; ddPCR; diagnosis; quantification; viral load.

FIG 1

SARS-CoV-2 detection process. (A) Generation of a reference sequence from a COVID-19 patient’s sample or by sequence alignment of publicly available sequences. The reference sequences can also be used to screen for emerging variants. (B) Target sites for developing RT-qPCR primers and probes, including targets commonly used by national public health institutions for RT-qPCR. (C) SARS-CoV-2 virion structure with locations of specific targets. (D) Common mutation spots associated with SARS-CoV-2 variants of concern.

FIG 2

dPCR workflow and principles of ddPCR and cdPCR. (A) SARS-CoV-2 sample collection processing. Arrows point to specific points where samples can be used for detection. Samples can be detected as crude lysates after inactivation, as purified RNA after extraction, or after RT-qPCR for further analysis. Ct, threshold cycle. (B) Droplet digital PCR workflow. (C) Chip/chamber-based dPCR workflow. dNTP, deoxynucleoside triphosphate.

FIG 3

Multiplex assay development using a two-color dPCR system. (A) General workflow for the detection of SARS-CoV-2 using dPCR. (B) Assay mix composition and dPCR workflow, including reaction mix preparation, partitioning, PCR amplification to the endpoint, and data analysis. cDNA, complementary DNA; dsDNA, double-stranded DNA. (C) Schematic representation of expected results per well from a two-color dPCR system when one (singleplex), two (2-plex), three (3-plex), or four (4-plex) targets are detected. T1 to -4, positive targets 1 to 4; Chl, channel; Neg, negative droplets.

FIG 4

SARS-CoV-2 environmental sample detection using RT-dPCR and determination of viable cells using propidium monoazide (PMA)-coupled RT-dPCR. (A to C) Wastewater (92–95) (A), surface (65, 90) (B), and aerosol (86, 87) (C) sample collection, processing, and detection. (D) PMA-coupled RT-dPCR for the determination of viable cells (98, 99). The PMA dye enters inactivated/dead cells with compromised membranes, and after light treatment, PMA covalently modifies the RNA. Subsequent amplification (PCR) of PMA-modified RNA templates is inhibited, while PMA-free RNA is amplified, enabling the selective quantification of RNA from viable cells. (The figure was constructed based on the information from the above-mentioned references.)

FIG 5

Applications of RT-dPCR in the diagnosis of COVID-19. (A) Diagnosis of RT-qPCR-negative patient samples, including patient discharge and follow-up. (B) COVID-19 patient viral load monitoring. (C) Pooled sample testing strategy to identify COVID-19 patients. (D) Resolving borderline RT-qPCR cases. (E) Development of commercial FDA EUA-authorized RT-dPCR diagnosis test kits.