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United States Department of Agriculture

Agricultural Research Service

Research Project: Detection and Management of Pathogens in Strawberry and Vegetable Production Systems

Location: Crop Improvement and Protection Research

Title: Rapid isothermal detection of Phytophthora species on plant samples using recombinase polymerase amplification

Authors
item Miles, Timothy
item Martin, Frank
item Coffey, Michael -

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 12, 2014
Publication Date: N/A

Interpretive Summary: New isothermal detection methods have been developed for the genus Phytophthora, an extremely important group of plant pathogens. Four assays were developed and were validated using purified DNA from many Phytophthora species and symptomatic plant samples collected throughout California. Results from these assays were similar to conventional methods. These techniques are rapid, sensitive, specific and do not require significant training to perform and offer many advantages over current technologies.

Technical Abstract: 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 Phytophthora have been developed which 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 Phytophthora genus-specific assay, two species-specific assays and a plant internal control. Assays were tested for sensitivity and specificity using DNA extracted from multiple plant species, 96 Phytophthora and 22 Pythium 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 Phytophthora 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.

Last Modified: 8/1/2014
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