A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.

Area of Science:

• Molecular Biology
• Virology
• Clinical Diagnostics

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since 2020.
• Accurate diagnosis of COVID-19 relies on detecting viral RNA via nucleic acid (NA) analysis.
• A global shortage of RNA extraction kits has impacted diagnostic capabilities.

Purpose of the Study:

• To develop a fast and cost-effective method for viral genome isolation for SARS-CoV-2 detection.
• To validate this method for sensitivity, specificity, linearity, reproducibility, and precision.
• To provide a solution to the reagent shortage affecting COVID-19 diagnostics.

Main Methods:

• A novel viral genome isolation protocol involving Proteinase K treatment and heat-shock incubation.
• Integration of the isolation method with a quantitative reverse transcription PCR (RT-qPCR) assay targeting the SARS-CoV-2 E gene.
• Validation using patient samples and comparison with automated extraction methods.

Main Results:

• The developed method demonstrated high sensitivity, detecting as low as 10 viral copies/sample.
• The workflow is rapid, significantly reducing analysis time compared to automated methods.
• The method achieved the same positivity rate as automated techniques while lowering costs.

Conclusions:

• This rapid viral genome isolation workflow is effective for SARS-CoV-2 RNA detection.
• The method offers a cost-effective and time-efficient alternative to existing extraction kits.
• The workflow is suitable for large-scale screenings and supports healthcare systems during outbreaks.