Genome guided LAMP assay for rapid and reliable detection of Xanthomonas axonopodis pv. vasculorum

Xanthomonas axonopodis pv. vasculorum (Xav), the causative agent of sugarcane gumming disease, represents a significant threat to global sugarcane production due to its systemic and destructive nature. A field-deployable tool specific to Xav is required for rapid infection detection and timely disease management. This resulted in a loop-mediated isothermal amplification (LAMP) assay targeting the gene region, unique to Xav strains, as a rapid and precise diagnostic assay. The selection of a target gene region was informed by comprehensive in silico genomes analyses of Xav and other closely related Xanthomonas species. The target gene region's specificity was validation against the NCBI GenBank database and internally sequenced genomes. Primers for both endpoint PCR and LAMP assays were designed using this unique gene. The LAMP assay underwent extensive testing against inclusivity and exclusivity panels. Use of exclusivity panel, comprising 81 strains from related species, other bacterial genera, and host genomes, demonstrated the assay's specificity with no false positives. The assay exhibited a detection limit of 1 pg, and its effectiveness was unimpeded by crude host lysate (sugarcane). Further validation through multi-device and multi-operator testing underscored the assay's 100% reproducibility and robustness. Application to infected plant samples resulted in the detection of all infected specimens without any false positives or negatives. This novel LAMP assay is accurate and reliable tool for Xav detection, with promising applications in routine diagnostics, biosecurity measures, microbial forensics, and epidemiological research.