Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

Area of Science:

• Molecular Diagnostics
• Virology
• Biotechnology

Background:

• The Bio-Rad SARS-CoV-2 ddPCR Kit was the first FDA-approved droplet digital PCR assay but lacked clinical validation.
• Droplet digital PCR (ddPCR) offers potential for enhanced sensitivity in detecting viral RNA.

Purpose of the Study:

• To clinically validate the Bio-Rad Triplex Probe ddPCR Assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection.
• To evaluate the ddPCR assay's performance in confirming weak-positive SARS-CoV-2 results.

Main Methods:

• Clinical validation of the Bio-Rad Triplex Probe ddPCR Assay.
• Determination of the limit of detection using serial dilutions of SARS-CoV-2 RNA.
• Comparison of ddPCR results with an FDA-validated RT-PCR assay on 48 positive and 30 negative specimens, plus 10 borderline cases.

Main Results:

• The limit of detection was 50 copies/mL (19/20 positive).
• The ddPCR assay demonstrated 97.9% positive percent agreement (47/48) and 100% negative percent agreement (30/30).
• Nine out of ten borderline positive cases were confirmed positive by ddPCR when tested in triplicate.

Conclusions:

• Droplet digital PCR (ddPCR) is an accurate method for SARS-CoV-2 detection with superior sensitivity for viral RNA.
• ddPCR can definitively evaluate borderline positive cases and suspected false-negative results.
• Clinical validation supports the use of ddPCR for reliable SARS-CoV-2 diagnostics.