A Rapid, Equipment-Free Method for Detecting Phytophthora infestans in the Field Using a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay

Abstract

Late blight, caused by the oomycete Phytophthora infestans, is a major constraint on the production of potatoes and tomatoes as well as a constant threat to global food security. An early diagnostic tool is important for the effective management of late blight in the field. Here, in combination with a simplified DNA extraction method, we developed a lateral flow strip-based recombinase polymerase amplification (LF-RPA) assay for the rapid, equipment-free detection of P. infestans. This assay targets the Ras-related protein (Ypt1) gene and can be performed over a wide range of temperatures (25 to 45°C). All 12 P. infestans isolates yielded positive detection results using the LF-RPA assay, and no cross-reaction occurred with related oomycetes or fungal species. With this assay, the detection limit was 500 fg of genomic DNA in optimized conditions. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire LF-RPA assay enabled the detection of P. infestans within 30 min with no specialized equipment. When applied to field samples, it successfully detected P. infestans in naturally diseased potato plants from eight different fields in China. Therefore, the LF-RPA assay is simple, rapid, and cost-effective and has potential for further development as a kit for diagnosing late blight in resource-limited settings or even on-site.