Field-Deployable Cross-Priming Amplification Coupled with Molecular Beacon Fluorometry for On-Site Detection of Citrus Huanglongbing Pathogen

Huanglongbing (HLB), a devastating citrus disease with no known cure, necessitates early on-site detection for effective disease management. However, current diagnostic methods remain limited by suboptimal detection rates, biosafety concerns, and lack of portability. This study presents the HLB-OSD system, a field-ready, portable heating and fluorometric device for the rapid, on-site identification of Candidatus Liberibacter asiaticus (CLas), the causative agent of HLB. The system integrates cross-priming amplification with highly specific molecular beacon probes in a single-tube, isothermal (63 °C) assay, enabling the direct detection of CLas from crude citrus leaf DNA extracts within 25-40 min without complex instrumentation. With 8.3 copy/μL detection limit and no cross-reactivity, the assay showed 94.24% concordance with qPCR (180/191). Field validation in Yongxiu county, a key HLB containment buffer zone in northern Jiangxi Province, confirmed a 9.0% (44/487) infection rate. Our on-site system provides lab-accurate detection, enabling timely monitoring of HLB for citrus protection.