One-Step RT-qPCR for Viral RNA Detection Using Digital Analysis

Area of Science:

• Virology
• Molecular Biology
• Biotechnology

Background:

• Rapid virus detection is crucial for preventing widespread infections.
• Current methods like RT-qPCR are time-consuming due to separate RNA extraction and cDNA synthesis steps.
• This limitation hinders rapid diagnosis and large-scale analysis needed for effective viral containment.

Purpose of the Study:

• To develop a simple and rapid thermolysis method for virus detection.
• To eliminate the need for viral RNA extraction and purification.
• To integrate this method with digital PCR for streamlined analysis.

Main Methods:

• A novel thermolysis protocol was developed to directly process viral samples.
• The protocol was integrated into a one-chip digital PCR (OCdPCR) system enabling thermolysis, RT, and digital PCR in a single unit.
• Optimization was performed using tobacco mosaic virus, followed by analysis of cucumber mosaic virus.

Main Results:

• Optimized thermolysis conditions were identified as 85°C for 5 minutes with a template concentration of 0.01 μg/nL.
• The integrated OCdPCR system successfully performed thermolysis, RT, and digital PCR in a single 30-minute reaction.
• The method achieved a quantification of 1,130.2 copies/µL for viral particles.

Conclusions:

• The developed thermolysis method significantly reduces the time required for viral RNA analysis.
• Integration with OCdPCR offers a streamlined workflow for rapid and precise viral quantification.
• This approach holds promise for future large-scale viral surveillance and diagnostics.