An Enzyme-Mediated Duplex Exponential Amplification Assay for Rapid Detection of Plasmodiophora brassicae

Clubroot, caused by the soilborne obligate biotrophic protist Plasmodiophora brassicae , substantially reduces the yield and quality of cruciferous crops worldwide. In this study, an enzyme-mediated duplex exponential amplification (EmDEA) assay was developed for rapid detection of P. brassicae in extracted DNA. By comparing the P. brassicae strain 3A genome with representative bacterial, fungal, and oomycete genome assemblies, we identified 157 candidate regions that showed no detectable matches in the selected non-target genomes under the specified alignment criteria. Pbspec1, a fragment within the annotated locus PBTT_08698, was selected based on computational non-target screening, within-species conservation, and suitability for assay design. PCR amplification and Sanger sequencing were used to validate the target fragment. Six forward primers, six reverse primers, and six RNA probes were screened, and F2–R4–RNA2 was selected for subsequent EmDEA experiments. Using serial dilutions of purified recombinant plasmid DNA, the F2–R4–RNA2 combination consistently detected template concentrations of 1.0 × 10 3 copies/μL and above. No amplification was observed from the 12 non-target fungal or bacterial organisms. The assay detected P. brassicae in DNA extracted from four infected root samples and three naturally infested soil samples. All tested positive samples produced detectable fluorescence during a programmed 20-min run on a portable isothermal fluorescence instrument. These results support the potential use of EmDEA for rapid preliminary screening of P. brassicae following DNA extraction.