Dual-Check CRISPR-SERS strategy for sensitively detecting Monkeypox DNA and its single-base mutated DNA

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nanotechnology

Background:

• Accurate and rapid detection of Monkeypox virus DNA is crucial for timely diagnosis and public health response.
• Existing detection methods may face limitations in sensitivity, speed, or adaptability to various genetic targets.
• The integration of CRISPR-Cas12a systems with nanotechnology offers potential for enhanced nucleic acid detection.

Purpose of the Study:

• To develop and validate a convenient and efficient dual-check detection strategy for Monkeypox DNA.
• To leverage the Surface-enhanced Raman Spectroscopy (SERS)-CRISPR-Cas12a system for high sensitivity and specificity.
• To demonstrate the adaptability of the system for detecting various nucleic acid sequences, including mutated variants.

Main Methods:

• Utilized a CRISPR-Cas12a system with a specific guide RNA (crRNA) to recognize Monkeypox plasmid DNA (mpDNA).
• Activated Cas12a's trans-cleavage activity to cleave Cy3-labeled DNA probes on gold nanoparticles (AuNPs).
• Quantified released Cy3 molecules using SERS on a silver nanopillar (AgNRs) substrate for signal amplification and detection.

Main Results:

• The SERS-CRISPR strategy successfully detected Monkeypox DNA across a concentration range from 5 nM to 0.5 fM.
• Achieved a limit of detection as low as 50 fM for Monkeypox DNA within a 60-minute timeframe.
• Demonstrated the capability to detect single-base mutated Monkeypox DNA, highlighting the system's specificity.

Conclusions:

• The developed SERS-CRISPR dual-check strategy provides a sensitive, rapid, and convenient method for Monkeypox DNA detection.
• The system's reliance on Cas12a's trans-cleavage activity allows for broad adaptability to detect diverse nucleic acid targets.
• This approach holds significant promise for advancements in molecular diagnostics and pathogen surveillance.