Ultrasensitive isothermal detection of a plant pathogen by using a gold nanoparticle-enhanced microcantilever sensor

Abstract

• GNP-enhanced dynamic microcantilever biosensor is constructed for pathogen analysis. • Combined with RPA, a LOD as low as one copy genomic DNA could be achieved. • This RPA-MCL could detect 57 ppm pathogen genomic DNA out of oilseed plant. Sensitive and rapid detection of plant pathogens, particularly for on-site diagnosis during custom inspections, is essential to prevent and control the spread of these pathogens. To address the need, a combined assay for detecting plant pathogens was developed by using gold nanoparticle-enhanced dynamic microcantilever (MCL) biosensing and isothermal recombinase polymerase amplification (RPA). In this study, we used Leptosphaeria maculans (L . maculans) , an aggressive fungus responsible for major losses in oilseed rape yield worldwide, as the target pathogen to demonstrate the merits of the combined RPA and MCL strategy (named as the RPA-MCL assay). The results indicated that the sensitivity of the RPA-MCL assay is higher than that of the fluorescence RPA assay, with the detection limit of only one copy of L . maculans DNA. In the practical assay, the newly developed RPA-MCL method was found to detect 57 ppm L . maculans genomic DNA in the oilseed rape seeds genomic DNA sample. Considering the high sensitivity and specificity of this strategy, we envision that the proposed RPA-MCL assay could have wide applications in nucleic acid diagnostics for on-site plant pathogen detection and species identification.