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

Area of Science:

• Agricultural diagnostics
• Molecular biology
• Nanotechnology

Background:

• Effective detection of plant pathogens like Avocado Sunblotch Viroid (ASBVd) is crucial for agricultural biosecurity.
• Current methods often require large sample volumes and lack the sensitivity for detecting low pathogen titers.
• Minimal sample collection is desirable for early disease detection and management.

Purpose of the Study:

• To develop a novel diagnostic platform for ultrasensitive and low-input detection of ASBVd.
• To enable rapid, field-deployable diagnostics for plant pathogens.
• To overcome limitations of existing diagnostic techniques in terms of sample volume and sensitivity.

Main Methods:

• Integration of isothermal recombinase polymerase amplification (RPA) with CRISPR-Cas12a detection.
• Utilization of a solid-state nanopore array for signal capture via pressure-driven blockage.
• Leveraging CRISPR-Cas12a collateral cleavage activity for single-bead fluorescent signal generation.

Main Results:

• Achieved a detection limit as low as 1.68 copies/μL for ASBVd.
• Required only 40 nL of bead-fluorophore mixture per readout, over 100-fold less than conventional assays.
• Demonstrated robust binary classification of ASBVd-positive and -negative samples from various avocado tissues and orchards.
• Assay completed in 60 minutes under isothermal conditions.

Conclusions:

• The developed platform offers ultrasensitive and low-input detection of ASBVd.
• The method is suitable for field deployment with minimal equipment, supporting on-site diagnostics.
• This technology holds promise for the detection of viroids and other low-titer pathogens in various settings.