Systematic development of species-specific assays for important Phytophthora spp. using recombinase polymerase amplification
T. D. MILES (1), F. N. Martin (2). (1) California State University Monterey Bay, Seaside, CA, U.S.A.; (2) USDA-ARS, Salinas, CA, U.S.A.
A platform for systematically developing rapid field-deployable isothermal assays for identification of <i>Phytophthora</i> at a genus and species specific level has been developed which focuses on the mitochondrial <i>atp9</i>-<i>nad9</i> locus utilizing a technique known as recombinase polymerase amplification (RPA). This approach was used to develop species-specific RPA assays for <i>P</i>. <i>cinnamomi</i>, <i>P</i>. <i>kernoviae</i>, <i>P</i>. <i>ramorum</i>, <i>P</i>. <i>rubi, </i>and<i> P</i>. <i>tentaculata</i>. These assays have been validated against 21 <i>Pythium</i> species, 1 <i>Phytopythium</i> sp. and over 100 <i>Phytophthora</i> species. Additionally, these assays were validated for sensitivity by diluting purified DNA from 10 ng down to 1fg. In general most RPA assays were capable of detecting the respective <i>Phytophthora</i> species between a range of 1pg-100 fg, which is similar to sensitivities with qPCR. When feasible, each assay was also validated with infected plant tissue collected in California. These RPA assays have many benefits over traditional diagnostic techniques because they don't require DNA extraction and can be run directly in the field in less than 25 min. Techniques for sequencing of amplified products have also been developed to confirm species identification.
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