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.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

“…The crude grapevine leaf-vein homogenates were tested with the validated LAMP assay, targeting BNp, and 23S rRNA LAMP assay targeting FDp (Kogovšek et al 2015) and the results compared to qPCR testing of extracted DNA. Additionally, the LAMP COX assay (Tomlinson et al 2010b) was used for confirmation of the capability of amplifying plant DNA in the sample. The analytical sensitivities of the on-site applicable leaf-vein crude homogenates testing were evaluated by testing five individual three-fold dilution series prepared from BNpinfected homogenates in healthy grapevine homogenate.…”

“…LAMP is a highly specific and rapid technique, and it also circumvents the sensitivity of PCR and qPCR to inhibitors in plant extracts (Francois et al 2011); furthermore, its isothermal nature provides the potential for it to be deployed in the field (Kogovšek et al 2015;Tomlinson et al 2010a). LAMP has shown a comparable or better performance to other detection methods and a wide applicability for the detection of plant pathogenic bacteria (Lenarčič et al 2014), viroids (Lenarčič et al 2013), fungi (Tomlinson et al 2010b) and phytoplasmas (Bekele et al 2011;Dickinson 2015;Hodgetts et al 2011;Kogovšek et al 2015;Tomlinson et al 2010a).…”

Use of Loop-Mediated Isothermal Amplification for Detection of Ophiostoma clavatum, the Primary Blue Stain Fungus Associated with Ips acuminatus

“…The aims of our study were therefore (i) to develop real-time LAMP assays for the species-specific detection of O. clavatum and O. brunneo-ciliatum directly in the insect vector, and an assay specific to I. acuminatus to be used as an internal control (36); (ii) to use these assays to assess which of these species is the blue stain fungus more consistently associated with I. acuminatus in the Southern Alps; and (iii) to investigate whether outbreak and nonoutbreak populations of I. acuminatus show significant differences in the occurrence of these symbiotic blue stain fungi.…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…LAMP method is very suitable for field testing and potentially valuable to laboratories without PCR facilities. This isothermal method has been applied for the rapid detection of Fusarium graminearum in contaminated wheat seeds (Abd-Elsalam et al, 2011) and for the detection of Phytophthora ramorum and P. kernoviae in field samples (Tomlinson et al, 2007(Tomlinson et al, , 2010.…”