Amplification-Free, Single-Microbead-Based Cas12a Assay for One-Step DNA Detection at the Single-Molecule Level

Area of Science:

• Biotechnology
• Molecular Biology
• Diagnostics

Background:

• CRISPR/Cas systems offer sensitive, specific DNA detection.
• Current CRISPR assays require nucleic acid amplification, increasing cost and complexity.

Purpose of the Study:

• To develop a one-step, amplification-free CRISPR/Cas12a DNA detection method.
• To achieve single-molecule detection sensitivity.

Main Methods:

• Combined CRISPR/Cas12a assay with a single-microbead detection platform.
• Utilized DNA reporters on biomimetic membrane-coated microbeads.
• Activated Cas12a cleaves reporters, causing bead luminescence.

Main Results:

• Achieved amplification-free, single-molecule DNA detection.
• Detected as few as three DNA copies in a 5 μL sample within 10 minutes.
• Successfully identified viral infections, foodborne bacteria, and DNA mutations in real samples.

Conclusions:

• The developed method enables rapid, sensitive, and specific DNA detection without amplification.
• Offers a novel approach for CRISPR/Cas-based DNA assays in biomedical applications.