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

• Published in: Phytopathology Vol. 100; no. 2; pp. 143 - 149
• Main Authors: Tomlinson, J.A, Dickinson, M.J, Boonham, N
• Format: Journal Article
• Language: English
• Published: St. Paul, MN American Phytopathological Society 01.02.2010
• Subjects: Bacterial plant pathogens; Biological and medical sciences; disease detection; DNA; DNA, Algal - genetics; DNA, Algal - isolation & purification; Electron Transport Complex IV - genetics; Fundamental and applied biological sciences. Psychology; host plants; immunologic techniques; loop-mediated isothermal amplification; methodology; microbial genetics; molecular sequence data; new methods; Nucleic Acid Amplification Techniques - methods; nucleotide sequences; pathogen identification; Phytopathology. Animal pests. Plant and forest protection; Phytophthora; Phytophthora - genetics; Phytophthora - isolation & purification; Phytophthora kernoviae; Phytophthora ramorum; Plant Diseases - microbiology; Quercus - microbiology; rapid methods; Rhododendron - microbiology; species differences; Stramenopila; Amplification; Plant pathology; Plant; Plant pathogen; Detection; Oomycota
• ISSN: 0031-949X; 1943-7684
• DOI: 10.1094/phyto-100-2-0143

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.