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

Abstract

Background Oak wilt disease, caused by Bretziella fagacearum is a significant threat to oak (Quercus spp.) tree health in the United States and eastern Canada. Without management the disease may cause dramatic changes to natural and urban ecosystems. Early and accurate diagnosis followed by timely treatment increases the level of disease control success. Results A rapid assay based on loop mediated isothermal amplification (LAMP) was first developed with fluorescence detection of the target fungus after 30-minute reaction time. Six different primers were designed to specifically bind and amplify the pathogen’s DNA. To simplify the assay for “point of care” (i.e., field) use, gold nanoparticles (AuNPs) were designed to bind to the amplified 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 sensitivity and specificity of the assay were calculated to be 100%. 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. Conclusions In summary, the LAMP assay coupled with oligonucleotide-conjugated gold nanoparticle visualization is a promising method for accurate and rapid molecular-based diagnosis in field settings. The new method can be adapted to other forest and plant diseases by simply designing new primers.