Rapid and sensitive one-pot CRISPR-Cas12a detection platform for common fungal species in ocular infections using multiplex crRNAs.

Corneal fungal infection is a major cause of visual impairment and can cause blindness if not diagnosed promptly. Traditional diagnostic methods are often time-consuming and require specialized expertise, UNDERSCORING the urgent need for rapid, sensitive, and user-friendly diagnostic tools. In this study, we utilized multiplex crRNAs in the Cas12a system to develop a highly sensitive and specific platform for the molecular detection of common fungal species causing corneal infections in a single reaction. We enhanced detection sensitivity through recombinase-aided amplification (RAA) and designed a streamlined tube with insert one-pot protocol to minimize aerosol contamination risk. Remarkably, our platform achieved a limit of detection of just 4 copies of fungal genomic DNA per reaction in only 32 min. It allows for direct detection from simulated tear samples, providing a potential for convenient and non-invasive testing option. Furthermore, our platform effectively tests clinical samples such as aqueous humor (AH) and vitreous humor (VH), demonstrating superior sensitivity compared to traditional PCR-based methods. These results highlight the potential of our platform as an on-site diagnostic solution for both clinical and non-clinical settings, contributing to the preservation of visual function.