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

Abstract

Late blight, caused by the oomycete <i>Phytophthora infestans</i>, 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 <i>P</i>. <i>infestans</i>. This assay targets the Ras-related protein (<i>Ypt1</i>) gene and can be performed over a wide range of temperatures (25 to 45°C). All 12 <i>P</i>. <i>infestans</i> 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 <i>P. infestans</i> within 30 min with no specialized equipment. When applied to field samples, it successfully detected <i>P</i>. <i>infestans</i> 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.