Recombinase polymerase amplification-lateral flow (RPA-LF) assay for rapid visual detection of Pseudomonas syringae pv. actinidiae in kiwifruit

Efficient detection of the causal pathogen Pseudomonas syringae pv. actinidiae (Psa) is vital to prevent and control Kiwifruit Bacterial Canker. In this present study, a novel isothermal amplification assay was developed for detecting Psa. The assay was highly specific for Psa, and could be completed in two steps within 35 min. Firstly, a set of RPA specific primers, which was designed based on the conserved region of avrD1 gene of Psa, was used to amplify a specific fragment of the avrD1 gene of Psa in a 30 min recombinase polymerase amplification (RPA) reaction. Secondly, lateral flow (LF) dipstick was used to detect and visualize the RPA amplicons of Psa within 5 min. An optimized detection system for Psa was established, with probe concentration of 0.10 μM, primer concentration of 0.42 μM, incubation time of 30 min and 5 min, and reaction temperature of 27–39 °C. The optimized RPA-LF method displayed high sensitivity to Psa, with 103 times higher sensitivity than that of conventional PCR, and could detect as low as 7.61 × 10−6 μg/μL genomic DNA of Psa in a 12.5 μL reaction system. Results in this study indicated that this rapid, specific, and sensitive RPA-LF assay has potential application prospects in detecting and monitoring of Kiwifruit Bacterial Canker, especially under limited time and resources.