A High-Sensitivity RPA/Cas12a Assay for Rapid Detection of the Select Agent Ralstonia solanacearum R3b2

Ralstonia solanacearum is a globally distributed, soil-borne species complex that causes bacterial wilt in many economically important crops. Race 3 biovar 2 (R3b2) is a cold-tolerant, highly aggressive subgroup within this complex that is responsible for an estimated $1 billion in annual global losses. Because R3b2 remains exotic to North America, it is classified as a USDA Select Agent due to its high potential to devastate U.S. solanaceous crops and ornamental industries. However, current diagnostic methods for this pathogen involve trade-offs among cost, sensitivity, specificity, and ease of use, particularly for point-of-care (POC) detection in field settings. CRISPR-based detection platforms have recently emerged as promising tools for high-sensitivity POC diagnostics. Here we developed an isothermal recombinase polymerase amplification (RPA)/Cas12a assay for specific, sensitive and rapid detection of R3b2. By targeting a genomic region associated with the cool-virulence phenotype, the assay reliably differentiates R3b2 from plant hosts and other diverse R. solanacearum strains. Incorporation of a dual-guide RNA system and an 8-mer single-stranded DNA (ssDNA) fluorescent reporter further improved sensitivity and reduced detection time. Using dilution series of R3b2-spiked Pelargonium DNA extracts, the assay detected as few as 4 CFU per reaction. To facilitate POC applications, the system was adapted into a one-pot, contamination-resistan¬t format compatible with portable fluorescence readers such as Genie III device and P51 viewer. By improving both sensitivity and portability, this assay enhances current diagnostic capabilities and, pending validation in real-world conditions, supports early surveillance efforts aimed at preventing the introduction of R3b2 into the United States.