A dual-modal RPA-CRISPR/Cas12a biosensor for rapid and ultrasensitive detection of Staphylococcus aureus in...

Bloodstream infections (BSIs) caused by Staphylococcus aureus (S. aureus) require rapid and accurate diagnosis to guide effective antimicrobial therapy and improve patient outcomes. However, current diagnostic methods often struggle to balance speed, sensitivity, portability, and cost.

After systematic optimization of reaction parameters (including primer design, reaction temperature and time, buffer composition, and probe concentration), the fluorescence assay demonstrated high specificity and completed detection within 35 min. Under optimized conditions (1.0 μM HS-ssDNA-MB and 10 min CRISPR incubation), the electrochemical sensor achieved a detection limit of 138 copies/mL, with a wide linear dynamic range from 4.37 × 10⁰ to 10⁵copies/μL, and showed high specificity against non-target pathogens. Furthermore, the platform demonstrated reliable performance in spiked artificial blood samples, with recovery rates ranging from 100.7% to 107.3%. This dual-modal recombinase polymerase amplification (RPA)-CRISPR/Cas12a biosensor combines the rapid amplification capability of isothermal methods with the high specificity of CRISPR-based detection.

This platform provides both a high-throughput fluorescence detection mode and a portable, low-cost electrochemical detection mode, offering an efficient and flexible solution for the rapid point-of-care diagnosis and large-scale screening of BSIs caused by S. aureus, with promising potential for clinical translation.