Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Accurate viral detection is crucial for pandemic management.
• CRISPR/Cas-based nucleic acid detection offers high sensitivity and specificity.
• Existing CRISPR methods often lack quantitative viral RNA measurement capabilities.

Purpose of the Study:

• To develop a quantitative RNA detection platform for viral RNA.
• To enhance CRISPR/Cas12b technology with digital reverse transcription loop-mediated isothermal amplification (RT-LAMP).
• To achieve absolute quantification of viral RNA with single-molecule sensitivity.

Main Methods:

• Development of a droplet digital reverse transcription loop-mediated isothermal amplification (RT-LAMP) enhanced Cas12b-based RNA detection platform (RECD).
• Integration of the thermally stable CRISPR/Cas12b system with digital RT-LAMP.
• Utilizing droplet digital technology for RNA detection and quantification.

Main Results:

• The developed droplet digital RECD (ddRECD) assay enables absolute quantification of viral RNA.
• The assay demonstrates single-molecule sensitivity.
• The platform leverages the digital format and CRISPR/Cas system for precise measurement.

Conclusions:

• The ddRECD assay provides a powerful tool for quantitative viral RNA detection.
• This method significantly advances molecular diagnostics for infectious diseases.
• The platform is expected to be valuable for pandemic control and research.