Recombinase Polymerase Amplification Assay for fast, sensitive and on-site detection of Phytophthora cactorum without DNA extraction

Authors: MUNAWAR, MUSTAFA - University Of Eastern Finland; TOLJAMO, ANNA - University Of Eastern Finland; Martin, Frank; KOKKO, HARRI - University Of Eastern Finland

Submitted to: European Journal of Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2018
Publication Date: 1/1/2019
Citation: Munawar, M., Toljamo, A., Martin, F.N., Kokko, H. 2019. Recombinase Polymerase Amplification Assay for fast, sensitive and on-site detection of Phytophthora cactorum without DNA extraction. European Journal of Horticultural Science. 84:14-19. https://doi.org/10.17660/eJHS.2019/84.1.2.
DOI: https://doi.org/10.17660/eJHS.2019/84.1.2

Interpretive Summary: This manuscript describes the development of a diagnostic assay using DNA based tools for rapid detection of a pathogen capable of causing significant losses of commercial strawberry plants. Its availability will simplify and speed up the diagnostics of this pathogen.

Technical Abstract: Crown rot, caused by Phytophthora cactorum, is an increasing problem for the strawberry crop in Europe. Most of the assays available for the detection P. cactorum are either laborious or inadequate for on-site testing. The isothermal Recombinase Polymerase Amplification (RPA) is an attractive alternative for rapid detection of pathogens from plant material. We have developed an RPA assay for P. cactorum using the intergenic mitochondrial DNA spacer between atp9 and nad9. The assay is specific, and sensitive with a lower limit of detection with purified DNA of 10 fg. Moreover the assay is DNA extraction-free, and requires only two minutes of tissue maceration prior to running the RPA assay.