Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids.

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Rapid diagnostics are critical for biopharmaceutical manufacturing and viral infection detection.
• Current methods like qPCR and CRISPR-based assays offer speed but lack precision due to variability.
• Digital PCR provides absolute quantification but is too slow for timely applications.

Purpose of the Study:

• To develop a rapid and accurate method for absolute quantification of nucleic acids.
• To address the limitations of existing diagnostic techniques in speed and variability.
• To introduce the RApid DIgital Crispr Approach (RADICA) for enhanced nucleic acid detection.

Main Methods:

• Development of the RApid DIgital Crispr Approach (RADICA).
• Utilized SARS-CoV-2 as a proof-of-concept target for validation.
• Tested RADICA's performance with Epstein-Barr virus (EBV) in various sample types.

Main Results:

• RADICA achieves absolute quantification of nucleic acids in 40-60 minutes.
• Demonstrated a wide linear dynamic range (0.6-2027 copies/μL for SARS-CoV-2, 0.5-2100 copies/μL for EBV) with high accuracy (R² > 0.99).
• Showcased high specificity, low variability, and tolerance to human DNA, outperforming digital PCR by 4-fold.

Conclusions:

• RADICA enables rapid, sensitive, and accurate absolute quantification of nucleic acids without thermal cycling.
• The method is versatile and validated for multiple targets, including viral DNA.
• RADICA presents a significant advancement for applications in clinical diagnostics, research, and biomanufacturing.