RPA-Assisted Cas12a System for Detecting Pathogenic Xanthomonas oryzae, a Causative Agent for Bacterial Leaf Blight Disease in Rice

Xanthomonas oryzae pv. oryzae (Xoo) is a widespread pathogen causing bacterial leaf blight (BLB) disease, devastating rice productivity in many cultivated areas of Thailand. A specific and simple method for Xoo detection is required to improve surveillance of disease transmission and outbreak. This study developed a recombinase polymerase amplification (RPA) assay assisted with CRISPR-cas12a assay (RAC) for Xoo detection from bacterial cell suspension of infected rice samples without DNA extraction. The efficiency of the RAC system for Xoo detection using either Xoo80 or Xoo4009 locus was optimized to amplify and determine the sensitivity and specificity using a Xoo DNA template from bacterial cell suspension of infected rice samples without DNA extraction. The RAC system using the Xoo4009 locus gave a higher specificity than Xoo80 locus, because only Xoo species was amplified positive RPA product with fluorescence signal by cas12a digestion, which indicated no cross reactivity. Optimal RAC using the Xoo4009 locus enabled diagnosis of Xoo presence from both plant extracted samples of Xoo artificially inoculated rice leaves within 3 d post-inoculation without symptomatic BLB appearance, and Xoo naturally infected rice. Findings exhibited that RAC using the Xoo4009 locus offered sensitivity, specificity and simplicity for Xoo detection, with low intensities of Xoo-DNA (1 x 103 copies/mu L) and Xoo-cell (2.5 x 103 cfu/mL). This developed RAC system showed significantly potential for Xoo detection at point-of-care application for early signs of BLB disease outbreak in rice fields.