Poster: Diseases of Plants: Disease Detection & Diagnosis
521-P
Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for Phytophthora ramorum.
J. BIENAPFL (1), L. Knight (1), Z. Abad (1) (1) USDA APHIS PPQ S&T, U.S.A.
Phytophthora ramorum was found as the causal agent of ‘Sudden Oak Death’ oaks and tanoaks in the coastal counties of California in the 1990s. Since then, the pathogen has been confirmed on numerous native hosts and nursery plants in California, Oregon, and Washington, with sporadic occurrences in other regions of the USA. The current methods of testing for P. ramorum include conventional and real-time PCR which are expensive and timely. The primary purpose of this work is to enhance Plant Protection and Quarantine efforts in the detection of P. ramorum by evaluating a field-deployable, simple, and rapid detection method. A previously published Recombinase Polymerase Amplification (RPA) Isothermal Amplification Assay for detection of P. ramorum was evaluated for both sensitivity and specificity using the TwistaTM apparatus. DNA extracted from pure culture, infected and healthy rhododendron tissue, as well as environmental samples were used to validate the P. ramorum protocol for regulatory use. Preliminary data comparing the isothermal amplification assay to current confirmatory P. ramorum diagnostic assays, evaluating ITS and Elicitin qPCRs shows that this protocol was not as sensitive as the ITS qPCR, but was comparable to the Elicitin qPCR. We will present the value of RPA isothermal amplification as a regulatory diagnostic tool for the rapid detection of P. ramorum.