Ultrasensitive, Low-Input Detection of Avocado Sunblotch Viroid via RPA-CRISPR and Nanopore-Array Single-Bead Fluorescence Readout

Abstract

Rapid and sensitive detection of plant pathogens, such as the Avocado Sunblotch Viroid (ASBVd), is essential for early disease management and agricultural biosecurity. Yet, most current diagnostic methods not only require relatively large sample inputs but also often lack the ultrasensitivity required for reliable detection with scarce or minimally collected plant material. Here, we report a novel low-input but ultrasensitive diagnostic platform that integrates isothermal recombinase polymerase amplification (RPA), CRISPR-Cas12a detection, and a solid-state nanopore array for the detection of ASBVd. The system leverages CRISPR-Cas12a collateral cleavage activity to generate single-bead fluorescent signals, which are captured by a nanopore array through pressure-driven blockage. Our platform achieves a detection limit down to 1.68 copies/L while using only 40 nL of bead-fluorophore mixture per readout, which is over 100-fold less than conventional assays based on fluorescent readout using an imaging reader, enabling detection from minimal avocado sample collection. We demonstrate robust binary classification of ASBVd-positive and -negative samples from multiple avocado tissue types and orchards in California. The assay requires just 60 minutes and operates entirely under isothermal conditions, avoiding the need for bulky PCR instruments and supporting on-site deployment with minimal equipment. This method provides a promising platform for field-deployable, ultrasensitive, and low-input diagnostics of viroids and other low-titer pathogens in plant or clinical settings.