The -CRISPR/Cas12a assay detected Pantoea stewartii subsp. stewartii at 1 × 10 ng DNA μL using multiple observation methods.
RPA-CRISPR/Cas12a assay demonstrates high sensitivity for detecting Pantoea stewartii subsp. stewartii.
The assay can identify the pathogen using real-time fluorescence, blue light observation, and lateral flow dipstick methods.
RPA-CRISPR/Cas13a assay detects Maize dwarf mosaic virus with an ultrasensitive limit of 3.69 × 10 ng μL.
Both assays effectively identify their respective pathogens without reducing speed or sensitivity.
These validated assays may serve as valuable tools for rapid detection of quarantine pathogens in maize.
Simplified
BACKGROUND: 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 () is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays.
RESULTS: RPA-CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1 × 10 ng DNA μLusing real-time fluorescence and blue light observation methods, and 1 × 10 ng DNA μLusing 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 ng μLusing the real-time fluorescence and blue light observation methods, and 3.69 × 10 ng μLusing the LFD. The dual RPA-CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays. -6 -1 -4 -1 -7 -1 -5 -1
