A Versatile CRISPR/Cas12a Autocatalytic Cascade System via Structure-Switching V-Type Split Probe for Highly...

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Accurate and rapid detection of pathogen nucleic acids is crucial for managing infectious diseases.
• Current molecular diagnostics, including CRISPR/Cas systems, often require preamplification, leading to complex workflows and expensive equipment.

Purpose of the Study:

• To develop a rapid, simple, and amplification-free CRISPR/Cas-based diagnostic system.
• To enhance sensitivity and reduce complexity in pathogen detection methods.

Main Methods:

• Utilized a structure-switching V-shaped DNA probe with a split Cas12a recognition sequence.
• Implemented a positive feedback loop and signal amplification cascade for exponential signal generation.
• Integrated the system into microfluidic and lateral flow assays.

Main Results:

• Achieved an ultralow background signal with rapid, exponential signal production.
• Demonstrated atto-molar sensitivity for pathogen DNA detection.
• Successfully applied to multiplex detection of human papillomavirus strains and point-of-care detection of monkeypox virus.

Conclusions:

• The developed amplification-free CRISPR/Cas system offers a significant advancement in rapid and sensitive pathogen DNA detection.
• This approach holds great potential for both clinical laboratory and point-of-care diagnostic applications.
• Simplifies workflows and reduces reliance on expensive equipment for infectious disease diagnostics.