Enhanced CRISPR/LbCas12a detection of Xanthomonas oryzae pv. oryzae using hairpin DNA probes coupled with RAA amplification for rapid field diagnosis of rice bacterial leaf blight

Rice bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating disease that threatens global rice security. Rapid and precise diagnosis of Xoo is important to control BLB and improve quality and quantity of rice production. This study reports a CRISPR/LbCas12a-based diagnostic platform that combines recombinase-aided amplification (RAA) with an engineered hairpin probe to enhance trans-cleavage activity. We hypothesize that the hairpin structure may enhance the accessibility or retention of the reporter within the activated LbCas12a complex, thereby improving trans-cleavage efficiency and fluorescence signal generation. The optimized assay demonstrated high sensitivity, detecting target DNA at concentrations as low as 100 aM and high specificity against related pathogens. Furthermore, the assay was successfully adapted to a lateral flow strip format, enabling rapid (20 min), visual, and on-site detection. When validated with field-collected samples, the results showed complete concordance with PCR. In summary, this work demonstrates enhanced LbCas12a trans-cleavage activity toward hairpin probes and provides a potential, highly sensitive tool for early BLB diagnosis, with promising applications in agricultural biosecurity.