Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the New Root-Lesion Nematode Pratylenchus dakotaensis on Soybean.

Root-lesion nematodes, Pratylenchus spp., are reported to cause serious yield losses in various crops, including soybean. A new root-lesion nematode species was detected in 2015 in a soybean field in North Dakota (ND) and named Pratylenchus dakotaensis in 2021. Nematode detection and differentiation from other species are critical in management strategies. Thus, a recombinase polymerase amplification (RPA) assay was developed for rapid detection of this nematode from field soils under isothermal conditions. New primers and probes were designed from internal transcribed spacer-ribosomal DNA region of the nematode genome and tested for both specificity and sensitivity. The RPA assay was able to detect DNA from a single adult nematode at 39.5°C in 20 min using both TwistAmp Basic and Exo Kits. The specificity of the primers was initially confirmed through in silico analyses and followed by laboratory tests. The assay successfully amplified DNA from the target species, although no amplification occurred for other Pratylenchus spp. and non-Pratylenchus control species. Sensitivity testing with real-time RPA revealed its ability to detect DNA in dilutions equivalent to 1/32 of a single nematode from DNA extracted from inoculated sterile soil. To further validate the assay, it was tested with 19 field soil samples collected in ND. This assay amplified soil DNA extracts of all P. dakotaensis-infested field samples confirmed through conventional PCR. It did not amplify DNA from 13 other field soils infested with other Pratylenchus spp. This is the first report of RPA development for detecting a root-lesion nematode species. The RPA assay developed can help in the rapid detection of this nematode species for effective nematode management.