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.
ABSTRACT 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.
Tomlinson
J A
JA
The Food and Environmental Research Agency, Sand Hutton, York YO41 1LZ, United Kingdom. jenny.tomlinson@fera.gsi.gov.uk
Dickinson
M J
MJ
Boonham
N
N
D048028:DNA, Algal / Q000235:genetics / Q000302:isolation & purification*; D003576:Electron Transport Complex IV / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010838:Phytophthora / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology*; D029963:Quercus / Q000382:microbiology; D029793:Rhododendron / Q000382:microbiology
D048028:DNA, Algal; D003576:Electron Transport Complex IV