Strategy to Develop and Evaluate a Multiplex RT-ddPCR in Response to SARS-CoV-2 Genomic Evolution

Area of Science:

• Virology
• Molecular Diagnostics
• Bioinformatics

Background:

• The emergence of SARS-CoV-2 necessitates accurate diagnostic tests to control viral spread.
• Virus evolution, including polymorphisms and mutations, can lead to false negatives in PCR-based diagnostics.
• Existing diagnostic methods struggle to keep pace with the diversity of evolving SARS-CoV-2 variants.

Purpose of the Study:

• To develop and evaluate a robust diagnostic methodology for SARS-CoV-2 detection that accounts for viral evolution.
• To assess the impact of viral mutations on PCR assay performance.
• To establish a reliable method for detecting SARS-CoV-2 in clinical and environmental samples.

Main Methods:

• In silico analysis of 154,489 whole-genome SARS-CoV-2 sequences to select PCR assay targets.
• Development of a duplex reverse-transcription droplet digital PCR (RT-ddPCR) assay.
• Experimental evaluation of assay sensitivity, specificity, and applicability using clinical and wastewater samples.

Main Results:

• The RT-ddPCR method was designed using comprehensive in silico analysis of global SARS-CoV-2 strains.
• The developed assays demonstrated high sensitivity and specificity in experimental evaluations.
• Preliminary results indicate the method's applicability for analyzing clinical and wastewater samples.

Conclusions:

• The proposed RT-ddPCR methodology offers a reliable approach for SARS-CoV-2 detection, adaptable to viral evolution.
• This method addresses the limitations of current diagnostics in identifying diverse and emerging SARS-CoV-2 variants.
• The study provides a validated tool for accurate SARS-CoV-2 surveillance in various sample types.