Vinni Thekkudan Novi, Hamada A Aboubakr, Melanie J Moore, Akli Zarouri, Jennifer Juzwik, Abdennour Abbas
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引用次数: 0
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. 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.
Results: 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.
Conclusions: 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.
背景：由 Bretziella fagacearum 引起的橡树枯萎病是对美国和加拿大东部橡树（栎属）健康的重大威胁。如果不加以控制，这种疾病可能会对自然和城市生态系统造成巨大破坏。早期准确诊断并及时治疗可提高疾病控制的成功率：结果：首先开发了一种基于环路介导等温扩增（LAMP）的快速检测方法，在 30 分钟的反应时间后用荧光检测 B. fagacearum。设计了六种不同的引物来特异性结合和扩增病原体的 DNA。为了简化该检测方法的现场使用，设计了金纳米粒子（AuNPs）来与 LAMP 反应得到的 DNA 扩增子结合。诱导沉淀后，AuNP-扩增子沉淀为肉眼可见的红色颗粒，表明病原体的存在。使用这种可视化方法对受感染的红橡树样本和健康样本进行了检测。结果发现，该检测方法对所研究的 B. fagacearum 分离物具有很高的诊断灵敏度和特异性。此外，所开发的检测方法还能在病变橡木样本的粗 DNA 提取物中检测病原体，这进一步缩短了处理样本所需的时间：总之，LAMP 检测法与寡核苷酸连接的金纳米粒子可视化相结合，是在野外环境中准确、快速地对法氏囊虫进行分子诊断的一种有前途的方法。只需设计新的引物，这种新方法就能适用于其他森林和植物疾病。
期刊介绍：
Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences.
There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics.
Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.
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