Rapid detection of Tomato brown rugose fruit virus (ToBRFV) using a CRISPR-Cas12a trans-cleavage fluorescence assay

Tomato brown rugose fruit virus (ToBRFV) has emerged as a significant threat to global tomato cultivation. While current diagnostic tools can detect ToBRFV, they are often expensive, technically demanding, and not easily adapted for use outside the laboratory. In this study, we developed a CRISPR-Cas12a trans-cleavage fluorescence assay integrated with PCR amplification for sensitive and specific detection of ToBRFV. The assay was developed using in silico-designed and chemically synthesised viral DNA templates, primers and CRISPR RNA (crRNA), enabling precise validation of Cas12a-mediated trans-cleavage activity. The use of a fluorophore-quencher (FQ) reporter allowed the direct visualisation of results under a portable blue/UV transilluminator. This PCR-Cas12a method demonstrated high sensitivity under the tested conditions and a faster turnaround, with visible detection possible in 30 min of Cas12a assay incubation following PCR amplification without the need for advanced equipment. This study highlights the advantages of Cas12a-based diagnostics and provides a foundation for developing rapid, efficient, and field-friendly assays for ToBRFV and other plant viruses.