Single and dual RPA-CRISPR/Cas assays for point-of-need detection of Stewart's wilt pathogen (Pantoea stewartii subsp. stewartii) of corn and Maize dwarf mosaic virus.

Abstract

Pantoea stewartii subsp. stewartii and Maize dwarf mosaic virus (MDMV) infections severely affect corn productivity worldwide. Rapid point-of-need diagnoses of quarantine pathogens P. stewartii subsp. stewartii and MDMV are required for early detection, timely disease management and ensuring phytosanitary regulations. Recombinase polymerase amplification (RPA) is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating CRISPR/Cas collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays. RPA-CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1 × 10-6 ng DNA μL-1 using real-time fluorescence and blue light observation methods, and 1 × 10-4 ng DNA μL-1 using the lateral flow dipstick (LFD). Likewise, RPA-CRISPR/Cas13a assay detected MDMV coat protein (CP) gene with an ultrasensitive detection limit of 3.69 × 10-7 ng μL-1 using the real-time fluorescence and blue light observation methods, and 3.69 × 10-5 ng μL-1 using the LFD. The dual RPA-CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays. The validated assays provide a useful and sensitive molecular tool for detecting two quarantine pathogens of maize within a minimal resource framework suitable for fast-tracking the containment strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.