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Rapid Detection of Phytophthora ramorum and P. kernoviae by Two-Minute DNA Extraction Followed by Isothermal Amplification and Amplicon Detection by Generic Lateral Flow Device

A method for nucleic-acid-based detection of pathogens in plant material has been developed which comprises a simple and rapid method for extracting DNA on the nitrocellulose membranes of lateral-flow devices, loop-mediated isothermal amplification (LAMP) of target DNA using labeled primers, and detection of the generically labeled amplification products by a sandwich immunoassay in a lateral-flow-device format. Each of these steps can be performed without specialist equipment and is suitable for on-site use, and a result can be obtained in just over an hour. A LAMP assay for the detection of plant DNA (cytochrome oxidase gene) can be used in conjunction with pathogen-specific assays to confirm negative results. The use of this method is demonstrated for the detection of Phytophthora ramorum, the causal agent of sudden oak death and dieback/leaf blight in a range of tree, shrub, and herbaceous species, and the recently described pathogen P. kernoviae.