A rapid LAMP assay for the diagnosis of oak wilt with the naked eye

Oak wilt disease, caused by Bretziella fagacearum is a significant threat to oak (Quercus spp.) tree health in the United States and Eastern Canada. The disease may cause dramatic damage to natural and urban ecosystems without management. Early and accurate diagnosis followed by timely treatment inc...

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Bibliographic Details
Published in:Plant methods 2024-08, Vol.20 (1), p.119-12, Article 119
Main Authors: Novi, Vinni Thekkudan, Aboubakr, Hamada A, Moore, Melanie J, Zarouri, Akli, Juzwik, Jennifer, Abbas, Abdennour
Format: Article
Language:English
Subjects:
Analysis
Bretziella Fagacearum
Diseases and pests
DNA
Ethylenediaminetetraacetic acid
Fluorescence
Gene amplification
Gold nanoparticles
LAMP assay
Methodology
Methods
Oak
Oak wilt
Red oak
Scientific equipment and supplies industry
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Summary:Oak wilt disease, caused by Bretziella fagacearum is a significant threat to oak (Quercus spp.) tree health in the United States and Eastern Canada. The disease may cause dramatic damage to natural and urban ecosystems without management. Early and accurate diagnosis followed by timely treatment increases the level of disease control success. A rapid assay based on loop mediated isothermal amplification (LAMP) was first developed with fluorescence detection of B. fagacearum after 30-minute reaction time. Six different primers were designed to specifically bind and amplify the pathogen's DNA. To simplify the use of this assay in the field, gold nanoparticles (AuNPs) were designed to bind to the DNA amplicon obtained from the LAMP reaction. Upon inducing precipitation, the AuNP-amplicons settle as a red pellet visible to the naked eye, indicative of pathogen presence. Both infected and healthy red oak samples were tested using this visualization method. The assay was found to have high diagnostic sensitivity and specificity for the B. fagacearum isolate studied. Moreover, the developed assay was able to detect the pathogen in crude DNA extracts of diseased oak wood samples, which further reduced the time required to process samples. In summary, the LAMP assay coupled with oligonucleotide-conjugated gold nanoparticle visualization is a promising method for accurate and rapid molecular-based diagnosis of B. fagacearum in field settings. The new method can be adapted to other forest and plant diseases by simply designing new primers.
ISSN:1746-4811
1746-4811
DOI:10.1186/s13007-024-01254-8