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

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 onsite testing. 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.