Integrated on-site detection of Fusarium temperatum based on a droplet digital CRISPR-based platform.

Fusarium temperatum (F. temperatum) is a fungus whose infection can cause various diseases in maize plants, leading to premature death, and F. temperatum mycotoxin poses a serious threat to human and animal health. Rapid and early on-site detection of F. temperatum infection facilitates the prevention of disease progression, which is an unmet need. In this study, a droplet digital CRISPR-Cas12a-based platform (DD-Cas), combined with a rapid extraction procedure, was developed for amplification-free on-site detection of F. temperatum. The DD-Cas assay can rapidly detect F. temperatum genomic DNA in infected maize samples with high sensitivity (102 CFU/mL) and specificity. Furthermore, we developed a smartphone-based fluorescence microscope integrating the heating module, that could accurately detect infected samples within 30 min, enabling low-cost point-of-care testing (POCT). This platform can avoid cross-contamination and amplification bias, thus having great potential for on field detection of pathogenic bacteria in agriculture.