A real-time recombinase polymerase amplification assay for the rapid detection of Vibrio harveyi.

摘要: Abstract Vibrio harveyi is a pathogen that infects fish and shellfish worldwide, causing severe economic losses for the aquaculture industry. As early diagnosis of V. infection crucial to disease surveillance prevention in cultured marine animals, fast accurate method detect required. Here, we performed recombinase polymerase amplification (RPA) using novel primers specifically designed recognize toxR gene, which encodes transmembrane protein, then hybridized this gene with carboxy fluorescein (FAM)-labeled probe. The optimal conditions real-time RPA assay were probe concentration 90 nM 20 min incubation at 37 °C. sensitivity our was 50 copies standard plasmid, while PCR 500 copies. In harveyi-spiked Pseudosciaena crocea samples, 60 CFUs per reaction, 600 CFUs reaction. SPSS probit regression analysis indicated limit detection (LOD) assay, 95% probability, 18 LOD reached within highly reproducible across eight independent assays. Our successfully differentiated from all other tested species, including some closely related. combination rapid DNA extraction protocol, tool monitoring outbreaks. This will be valuable