P-203 - Rapid Multiplex detection of Verticillium dahliae and Pratylenchus penetrans causing Potato Early Dying Disease using RPA-CRISPR/Cas12a assay

Potato early dying (PED) disease complex threatens potato production worldwide, reducing up to 50% of the potato yield. PED is caused by the soilborne fungus Verticillium dahliae and the root lesion nematode Pratylenchus penetrans. Early and accurate detection of both pathogens is critical for timely disease management. Conventional diagnostic methods, such as PCR, qPCR, and ddPCR, rely on separate identification of each pathogen, are complex, time-consuming, and require a well-equipped laboratory. The study aims to develop a single diagnostic assay for the rapid detection of PED pathogens. Assay development included the combination of recombinase polymerase amplification (RPA) with regularly clustered interspaced short palindromic repeats (CRISPR). The conserved genomic regions, including the intergenic spacer (IGS) of V. dahliae and the cytochrome oxidase I (COI) gene of P. penetrans, were targeted to design specific primer pairs and guide RNAs. RPA requires minimal sample preparation, constant
low temperature (37-40 ⁰C), and short duration (20-30 minutes). Subsequent Cas12a cleavage, guided by the pathogen-specific guide RNAs, produces a fluorescent signal that is detected either with a portable thermal cycler or fluorescence reader. This streamlined workflow significantly reduces sample detection time to under 90 minutes compared to conventional diagnostic assays while lowering specific laboratory equipment. This assay's high sensitivity and specificity would offer a robust, multiplex platform for rapid and accurate pathogen surveillance in potato production systems.