CRISPR-Cas13-based amplification-free detection of three quarantine-significant sugarcane viruses.

Crop losses from viral pathogens cost billions of dollars annually, and sugarcane is particularly vulnerable. Sugarcane is the most cultivated crop in the world by volume; therefore, significant yield losses result in large dollar losses for farmers and trade economies. In some cases, viruses contribute to more than half of the yield reduction. Three quarantine-significant pathogens-Sugarcane mosaic virus (ScMV), Sugarcane streak mosaic virus (ScSMV), and Sugarcane yellow leaf virus (ScYLV)- pose major threats to global sugarcane production because of their rapid spread, asymptomatic infections, and delayed symptom expression. Effective management of these pathogens requires robust, rapid, and accurate detection methods that can distinguish among these viruses, even at low titers. Here, we report the development of an amplification-free CRISPR-Cas13-based assay with a limit of detection (LOD) of ~1.5 pM. The assay allows for simultaneous screening of all three viruses directly from bulk RNA extracts of plant material. Our approach leverages LwaCas13a specificity to detect and differentiate viral targets in a single assay. Compared with conventional multi-step assays, this strategy significantly reduces time and contamination risk. Validation on naturally infected material demonstrated its sensitivity and reliability. This platform provides a powerful tool for early and accurate diagnosis of ScMV, ScSMV, and ScYLV, with strong potential for integration into sugarcane virus surveillance and management programs.