Development of a rapid, pan fungal diagnostic assay

摘要 诊断真菌感染面临一系列挑战，包括 比较鉴定真菌和减少真菌鉴定专用仪器和化验的数量 细菌和病毒。这些问题因真菌感染患者是 通常是免疫抑制，这使他们容易受到常见和罕见真菌的感染。 从历史上看，侵袭性感染的分子诊断分析一直是劳动密集型的，需要很高的 技术专长的程度。开发一种高效的检测方法，可以用技术来替代 传统的真菌学专业知识一直很难掌握。而核糖体的ITS测序是真菌的黄金标准 分子诊断速度很慢，需要很高的资金成本，而且对许多真菌来说，还不够具体。这 这项研究将依赖于两个创新组件，这将使几乎任何真菌能够识别到 迅速达到物种水平。第一个组成部分是一种名为纳米孔测序的新测序技术， 这是快速和廉价的。第二部分将由一种新的测序目标组成，称为 基因间隔区序列(IGS)，位于真菌核糖体重复序列内。IGS太长了，无法 在诊断分析中被传统的Sanger测序所覆盖，因为从一个 因为需要走引子，所以不能用于真菌鉴定。然而，纳米孔测序可以 只需一次测序即可轻松覆盖IGS区域。实现发展高效PAN的目标 真菌诊断策略，三个目的将检验测序的假设，使用较少，但高度 使用纳米孔测序的真菌rDNA重复基因的信息区将提供最大 真菌鉴定的特异性和迄今为止最快的周转时间。这些目标是：1)生成 GenBank RefSeq中的真菌基因间隔区序列参考序列数据库(参考文献 序列)靶向基因座计划，称为真菌IGS RefSeq数据库2)，快速分析的发展 用于基于基因间隔区序列的纳米孔测序的真菌鉴定，3)比较 针对当前实验室真菌诊断方法的纳米孔序列诊断分析。长期的 这个项目建成后的目标将是未来开发一个快速、廉价、 在能力上是泛真菌的化验。

ABSTRACT Diagnosing fungal infections faces a number of challenges including a decline in expertise needed for identifying fungi and a reduced number of instruments and assays specific for fungal identification compared to bacteria and viruses. These problems are exacerbated by the fact that patients with fungal infections are often immunosuppressed, which predisposes them to infections from both common and rarely seen fungi. Historically, molecular diagnostic assays for invasive infections have been labor intensive and required a high degree of technical expertise. Developing an efficient assay that could use technology as a substitute for classical mycology expertise has been difficult. While ribosomal ITS sequencing is the gold standard of fungal molecular diagnostics, it is slow, requires high capital costs, and for many fungi, is not specific enough. This study will rely on two innovative components that will enable the identification of almost any fungus to the species level quickly. The first component is a new sequencing technology called nanopore sequencing, which is fast and inexpensive. The second component will consist of a novel sequencing target called the intergenic spacer sequence (IGS), which is located within the fungal ribosomal repeat. The IGS is too long to be covered by traditional Sanger sequencing in a diagnostic assay because multiple reads generated from a primer walk would be required, so it is not used for fungal identification. However, nanopore sequencing can easily cover the IGS region in a single sequencing run. To achieve the goal of developing an efficient pan fungal diagnostic strategy, three aims will test the hypothesis that sequencing a little used, but highly informative region of the fungal rDNA repeat locus using nanopore sequencing will provide the greatest specificity and quickest turnaround time to date for fungal identification. These aims are: 1) Generation of a fungal intergenic spacer sequence reference sequence database within the GenBank RefSeq (reference sequence) Targeted Loci Project, called the Fungal IGS RefSeq database 2), Development of a rapid assay for fungal identification based on nanopore sequencing of the intergenic spacer sequence, 3) Comparison of a nanopore sequence diagnostic assay against current laboratory fungal diagnostic methods. The long-term goal that this project will enable after its completion will be the future development of a rapid, inexpensive, assay that is pan fungal in capability.