On-site, highly-sensitive and multi-signal detection of cucumber bacteria via CRISPR/Cas12a coupled with duplex RPA

Pseudomonas amygdali pv. lachrymans (Pal) and Pectobacterium brasiliense (Pb) are highly destructive bacterial pathogens that cause severe oozy diseases in cucumber crops, often resulting in significant yield losses. Rapid and accurate detection is essential for effective disease management, yet current molecular detection methods are limited by their reliance on sophisticated instrumentation, time-consuming procedures, and laboratory-dependent operations, significantly limiting their practicality in field settings. To address these limitations, we developed a portable detection platform that integrates dual recombinase polymerase amplification with CRISPR/Cas12a technology. This system demonstrated exceptional sensitivity with a detection limit of 10⁻⁵ ng/μL for both Pal and Pb, while exhibiting high specificity and enabling multi-signal output capabilities. For rapid on-site detection, we further developed a portable device integrating a heating unit and a blue-light-excited fluorescence detection system, coupled with a smartphone-based readout for high-throughput field testing. The entire process was completed within one hour. This platform not only provides a powerful tool for rapid field detection of plant pathogens, but also pioneers a novel universal nucleic acid detection strategy with broad application prospects in point-of-care molecular diagnostics across healthcare, agriculture and other fields. By bridging the gap between laboratory precision and field practicality, this technology opens new avenues for decentralized diagnostics in crop protection, microbial ecology, and public health.