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

Aug 20, 2024Biosensors & bioelectronics

Highly sensitive detection of Staphylococcus aureus using a DNA-cutting system combined with CRISPR/Cas12a

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Abstract

A detection method for S. aureus achieves a limit of detection of 5 CFU/mL in 29 minutes without nucleic acid extraction.

The CRISPR/Cas12a system can be activated by manganese ions after forming a complex with specific aptamers and nanoparticles.
The proposed strategy eliminates the need for nucleic acid amplification, which can reduce contamination risks.
The method demonstrated ultrasensitive quantitative detection of S. aureus, indicating potential effectiveness for food safety applications.
The detection approach may be adaptable to other microorganisms by modifying the recognition element.

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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. NH-modified S. aureus-specific aptamer was loaded on the surface of FeOMNPs (apt-FeOMNPs) and MnONPs (apt-MnONPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-FeOMNPs/S. aureus/apt-MnONPs complex and then MnONPs were etched to release Mnto 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 Mnthrough 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. 2+ 2+ 2+ 2 3 4 3 4 2 2 3 4 2 2

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

Abstract

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