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

The stubby root nematode, is one of the most important plant-parasitic nematodes. Besides root feeding, also transmits the in potatoes, which causes corky ringspot disease. Rapid detection of is key for efficient management. This study was conducted to develop a real-time recombinase polymerase amplification (RPA) assay that is capable of detecting 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 and was used along with primers designed previously. The real-time RPA assay had the ability to detect 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 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 detection directly from soil DNA using real-time RPA and is the fastest method for detection in soil to date.