Ultrasensitive detection platform for Staphylococcus aureus based on DNAzyme tandem blocking CRISPR/Cas12a system

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Food Safety

Background:

• CRISPR/Cas12a systems are vital for detecting pathogens but often require amplification, increasing time and contamination risk.
• Current methods for sensitive pathogen detection face challenges with speed and complexity.

Purpose of the Study:

• To develop a rapid, sensitive, and amplification-free detection strategy for Staphylococcus aureus (S. aureus) using CRISPR/Cas12a and DNAzyme.
• To overcome limitations of existing CRISPR-based detection methods, such as extended detection times and nucleic acid contamination.

Main Methods:

• A modified CRISPR/Cas12a system with blocked Cas12a activity (bcrRNA) was employed, reactivated by Mn2+ released from etched MnO2 nanoparticles.
• S. aureus-specific aptamers functionalized magnetic (Fe3O4) and MnO2 nanoparticles captured the target bacteria.
• DNAzyme activity, triggered by Mn2+, cleaved bcrRNA, restoring CRISPR/Cas12a function for detection.

Main Results:

• The assay achieved ultrasensitive quantitative detection of S. aureus with a limit of detection (LOD) of 5 CFU/mL.
• The entire detection process was completed rapidly within 29 minutes.
• The method demonstrated high performance without requiring nucleic acid extraction or amplification.

Conclusions:

• The developed CRISPR/Cas12a and DNAzyme strategy offers a rapid and sensitive platform for S. aureus detection.
• This amplification-free approach enhances food safety and public health diagnostics by reducing time and contamination risks.
• The platform's adaptability suggests potential for developing a universal detection strategy for various targets.