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

Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn ²⁺ . NH ₂ -modified S. aureus-specific aptamer was loaded on the surface of Fe ₃ O ₄ MNPs (apt-Fe ₃ O ₄ MNPs) and MnO ₂ NPs (apt-MnO ₂ NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe ₃ O ₄ MNPs/S. aureus/apt-MnO ₂ NPs complex and then MnO ₂ NPs were etched to release Mn ²⁺ to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn ²⁺ through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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