Rapid and Direct Detection of the Stubby Root Nematode, Paratrichodorus allius, from Soil DNA Extracts Using Recombinase Polymerase Amplification Assay

The stubby root nematode, <i>Paratrichodorus allius,</i> is one of the most important plant-parasitic nematodes. Besides root feeding, <i>P. allius</i> also transmits the <i>Tobacco rattle virus</i> in potatoes, which causes corky ringspot disease. Rapid detection of <i>P. allius</i> is key for efficient management. This study was conducted to develop a real-time recombinase polymerase amplification (RPA) assay that is capable of detecting <i>P. allius</i> directly in DNA extracts from soil using a simple portable device in real time. A fluorophore-attached probe was designed to target the internal transcribed spacer (ITS)-rDNA of <i>P. allius</i> and was used along with primers designed previously. The real-time RPA assay had the ability to detect <i>P. allius</i> DNA extracted directly from infested soil with a sensitivity of one-sixteenth portion of a single nematode. This RPA assay was specific, as it did not produce positive signals from non-target nematodes tested. The real-time RPA was found to be rapid as it could even detect <i>P. allius</i> in as little as 7 min. Testing with 15 field soil samples validated the RPA assay developed in this study. This is the first report of <i>P. allius</i> detection directly from soil DNA using real-time RPA and is the fastest method for <i>P. allius</i> detection in soil to date.