Location: Crop Improvement and Protection ResearchTitle: Development of rapid isothermal amplification assays for Phytophthora species from plant tissue) Author
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2014
Publication Date: 1/15/2015
Citation: Miles, T.D., Martin, F.N., Coffey, M. 2015. Development of rapid isothermal amplification assays for Phytophthora species from plant tissue. Phytopathology. 105:265-278. Interpretive Summary: Having a rapid, DNA based, pathogen detection technique that could be used directly in the field would facilitate management of diseases, regulatory concerns on movement of pathogens, and provide a useful tool for researchers. This manuscript describes a molecular technique called isothermal amplification (amplification is done at a constant temperature) for detection of Phytophthora spp. at a genus and species specific level that does not require DNA extraction, can be completed in as little as 15-20 minutes directly in the field using an inexpensive portable hand held unit, and is nearly as sensitive as more elaborate laboratory assays requiring highly purified DNA and expensive laboratory equipment. Techniques also have been developed for sequencing amplified products to determine what species they are, thereby reducing the need for pathogen culturing.
Technical Abstract: Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real time PCR for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid techniques for detection of Phytophthora, one genus specific assay multiplexed with a plant internal control and the other a species-specific assay (P. ramorum and P. kernoviae). Assays were tested for sensitivity (ranging from 10 ng to 1 fg of DNA) and specificity using DNA extracted from more than 96 Phytophthora, 21 Pythium, 1 Phytopythium and a wide range of plant species. The lower limit of linear detection using purified DNA was 1 pg of DNA 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 by collecting 222 symptomatic plant samples from over 50 different hosts. Ninety-one samples were positive using the Phytophthora genus specific RPA test and the same samples were also positive using TaqMan real time PCR, but cultures were obtained for only 62% of these samples. A technique for the generation of sequencing templates from positive samples to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 minutes), do not require DNA extraction or extensive training to complete, use less expensive equipment than PCR based assays and are significantly more specific than current immunologically based methods. The ability to sequence amplified templates also allows for identification of the species present without the need for culturing.