Development and Analytical Validation of a One-Step Five-Plex RT-ddPCR Assay for the Quantification of SARS-CoV-2...

Area of Science:

• Molecular Biology
• Virology
• Infectious Diseases

Background:

• Sensitive SARS-CoV-2 detection is crucial for controlling the COVID-19 pandemic.
• Existing methods require optimization for sensitivity and standardization.
• The nucleoprotein (N) gene is a key target for SARS-CoV-2 diagnostics.

Purpose of the Study:

• To develop and validate a highly sensitive five-plex one-step RT-ddPCR assay for SARS-CoV-2.
• To enable simultaneous absolute quantification of viral targets and internal controls.
• To improve standardization of molecular testing for SARS-CoV-2.

Main Methods:

• In-silico design of novel primers and probes for three SARS-CoV-2 N gene regions (N1, N2, N3).
• Incorporation of synthetic RNA external control (RNA-EC) and Beta-2-Microglobulin (B2M) internal control (RNA-IC).
• Analytical validation using synthetic standards and comparison with a commercial CE-IVD RT-qPCR assay on 100 clinical specimens.

Main Results:

• The assay demonstrated high analytical sensitivity, specificity, linearity, reproducibility, and recovery rates, even at low viral loads.
• Simultaneous quantification of RNA-EC and RNA-IC ensured robust quality control.
• The RT-ddPCR assay showed high concordance (99%) and superior sensitivity compared to the RT-qPCR assay.

Conclusions:

• The developed five-plex one-step RT-ddPCR assay provides sensitive, specific, and reproducible absolute quantification of SARS-CoV-2.
• The inclusion of external and internal RNA controls enhances standardization for clinical and wastewater testing.
• This assay is a valuable tool for accurate and reliable SARS-CoV-2 molecular diagnostics.