An Ultra‐Sensitive Quarantine Pathogen On‐Site Detection Based on a One‐Pot Asymmetric Recombinase Polymerase Amplification and MNAzyme ‐Assisted Target Recycling Biosensor ( OAR ‐ MNA )

ABSTRACT The dissemination of quarantine pathogens poses a significant risk to global crop production, threatening crop health and disrupting agroecosystems. Timely and accurate on‐site detection is crucial to prevent outbreaks. Recombinase polymerase amplification (RPA) and multicomponent DNAzyme (MNAzyme) are promising isothermal detection technologies; however, their integration has been hindered by the requirement of single‐stranded DNA (ssDNA) for MNAzyme activation, as RPA generates double‐stranded DNA (dsDNA). To address this, we developed the OAR‐MNA biosensor, an innovative one‐pot platform coupling asymmetric RPA (aRPA) with MNAzyme‐assisted target recycling. The biosensor was applied to detect Acidovorax citrulli , the causative agent of bacterial fruit blotch (BFB) in cucurbits. By adjusting RPA primer concentrations, aRPA generates abundant ssDNA, enabling visual detection of A. citrulli with a detection limit of 20 copies/μL and perfect specificity, surpassing the performance of fluorescent RPA. The biosensor demonstrated 100% concordance with qPCR in seed samples and maintained robust performance in spiked melon homogenate. Our comparative analysis revealed advantages of the OAR‐MNA system, including reduced detection costs and enhanced field applicability. When coupled with a portable DNA analyser, the biosensor provides user‐friendly results (+/−), making it ideal for on‐site deployment. Beyond BFB detection, OAR‐MNA has been successfully applied to the detection of Cucumber green mottle mosaic virus (CGMMV), an RNA quarantine pathogen that causes severe losses to cucurbit crop production. By enabling early, accurate on‐site diagnosis, the OAR‐MNA biosensor represents an advancement in plant pathogen detection technology, offering a promising tool for safeguarding global crop production.