Rapid isothermal detection and species-specific assay of Phytophthora in plant samples using recombinase polymerase amplification
T. D. MILES (1), F. N. Martin (2), M. D. Coffey (3). (1) USDA/ARS, East Lansing, CA, U.S.A.; (2) USDA/ARS, Salinas, CA, U.S.A.; (3) University of California - Riverside, Riverside, CA, U.S.A.
Recently several isothermal amplification techniques have been developed that are extremely tolerant towards inhibitors present in many plant extracts. Recombinase polymerase amplification (RPA) assays for the genus <i>Phytophthora</i> have been developed to provide a simple and rapid method to macerate plant tissue and detect target DNA using primers and a labeled probe. Four RPA assays were developed, a <i>Phytophthora</i> genus-specific assay, <i>P</i>. <i>ramorum</i> and <i>P</i>. <i>kernoviae</i> species-specific assays and a plant internal control. Assays were tested for sensitivity and specificity using DNA extracted from multiple plant species, 96 <i>Phytophthora</i> and 22 <i>Pythium </i>spp. The lower limit of linear detection using purified DNA was 1 pg in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field with 222 symptomatic plant samples from over 51 different hosts throughout California. Ninety-one samples were positive using the <i>Phytophthora</i> genus specific RPA test and the same samples were also positive using TaqMan PCR and traditional isolation techniques. A technique for the generation of sequencing templates from positive samples to confirm species identification has been developed. These RPA assays have added benefits over traditional technologies because they run in less than 25 min, don't require DNA extraction, are field portable and are more specific than current immunologically based methods.
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