Sensitive Detection of Ralstonia solanacearum Using Recombinase-Aided Amplification and an Aerolysin Nanopore

Ralstonia solanacearum is a soil-borne plant pathogenic bacterium that causes bacterial wilt disease, leading to substantial economic losses in over 250 crops, including tomatoes, tobacco, and potatoes. We developed a novel method for the specific detection of R. solanacearum by combining recombinase-aided amplification (RAA) with wild-type aerolysin nanopore. The probe Target-16 was hybridized to one strand of the RAA product, allowing the nicking endonuclease Nb.BsrDI to recognize specific cleavage sites and cleave the product into two short DNA strands. As these short DNA strands passed through the aerolysin nanopore, they produced distinct current-blockage signals that were markedly different from those generated by Target-16, thereby enabling on-site detection of R. solanacearum. This method revealed high sensitivity (10 pg/μL) and specificity and reproducibility. The strategy is less dependent on expensive instruments and pure DNA, and can be performed within 3.2 h at constant temperature, which can be utilized to detect R. solanacearum in tobacco samples, yielding results consistent with those obtained using PCR and DNA sequencing. This method provides a powerful tool for the rapid detection of R. solanacearum, especially suited for fieldwork or laboratories with limited resources.