Development of a rapid, pan fungal diagnostic assay

摘要 诊断真菌感染面临着许多挑战，包括对真菌感染所需的专业知识的下降。 鉴定真菌和减少数量的仪器和测定具体的真菌鉴定相比， 细菌和病毒。这些问题因真菌感染患者的 通常免疫抑制，这使他们容易受到常见和罕见真菌的感染。 从历史上看，用于侵入性感染的分子诊断测定一直是劳动密集型的，并且需要较高的成本。 技术专长的程度。开发一种有效的检测方法，可以使用技术作为替代品， 传统的真菌学专业知识是很困难的。虽然核糖体ITS测序是真菌鉴定的金标准， 分子诊断是缓慢的，需要高的资本成本，并且对于许多真菌，不够特异。这 这项研究将依赖于两个创新的组成部分，这将使几乎任何真菌的鉴定， 物种的迅速发展。第一个组成部分是一种新的测序技术，称为纳米孔测序， 这是快速和廉价的。第二个组成部分将包括一个新的测序靶点，称为 基因间间隔序列（IGS），其位于真菌核糖体重复序列内。IGS太长， 在诊断测定中，传统的桑格测序覆盖了多个读段， 需要引物步移，因此不用于真菌鉴定。然而，纳米孔测序可以 在单次测序运行中轻松覆盖IGS区域。为了实现开发高效锅的目标， 真菌诊断策略，三个目的将测试的假设，测序一点用，但高度 使用纳米孔测序的真菌rDNA重复基因座的信息区域将提供最大的 特异性和最快的周转时间。这些目标是：（1）产生一个 GenBank RefSeq中的真菌基因间间隔序列参考序列数据库（参考 靶向基因座项目，称为真菌IGS RefSeq数据库2），快速测定的开发 用于基于基因间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测定。长期 该项目完成后的目标将是未来开发一种快速，廉价， 在能力上是泛真菌的测定。

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.