Split G-Quadruplex-Integrated CRISPR-Cas Biosensor for One-Pot, Signal-On Visual Detection of Pathogen Nucleic Acids

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Pathogen nucleic acid analysis is crucial for clinical management and disease surveillance.
• Existing methods can be time-consuming and require complex laboratory setups.
• There is a need for rapid, accurate, and accessible diagnostic tools for infectious diseases.

Purpose of the Study:

• To develop a novel, one-pot, colorimetric biosensing platform for pathogen nucleic acid detection.
• To integrate G-quadruplex structures with CRISPR-Cas systems for enhanced sensitivity and specificity.
• To create a user-friendly system suitable for point-of-care applications and real-time surveillance.

Main Methods:

• A G-quadruplex-integrated CRISPR-Cas biosensing platform utilizing a signal-on mechanism.
• Development of a universal visual probe for colorimetric detection coupled with Cas12 and Cas13 systems.
• Integration of recombinase polymerase amplification (RPA) with CRISPR-mediated cleavage in a tube-in-tube cartridge system.
• Validation using monkeypox virus (MPXV) and respiratory syncytial virus (RSV) as model targets.

Main Results:

• The platform demonstrated a distinct green colorimetric signal upon target recognition.
• Achieved a detection limit of 1 copy per test for MPXV and RSV.
• Showcased perfect concordance with PCR methods (40/40 agreement).
• Validated for detecting MPXV in environmental samples and RSV in clinical specimens.

Conclusions:

• The developed G-quadruplex-integrated CRISPR-Cas biosensing platform offers rapid (<60 min) and accurate pathogen detection.
• The one-pot, colorimetric system eliminates cross-contamination risks and simplifies workflow.
• This novel molecular diagnostic paradigm is suitable for real-time surveillance and point-of-care applications.