Development of a rapid, pan fungal diagnostic assay

快速、泛真菌诊断测定的开发

• 批准号: 10552690
• 负责人: BRIAN WICKES
• 金额: $ 45.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552690
• 关键词: Aspergillus; Bacteria; Base Sequence; Biological Assay; C10; Candida; Candida auris; Capital; Case Study; Cessation of life; Clinical; Computers; Contracts; Cryptococcus; Dedications; Deposition; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Dideoxy Chain Termination DNA Sequencing; Disease Outbreaks; Drug resistance; Epidural Injections; Equipment; Etiology; FDA approved; Face; Failure; Foundations; Frequencies; Future; Genbank; Generations; Goals; Healthcare; Hour; Human; Immune system; Immunocompromised Host; Infection; Laboratories; Literature; Maintenance; Malaria; Microbiology; Missouri; Molds; Molecular; Molecular Diagnostic Testing; Morbidity - disease rate; Multi-Drug Resistance; Mycoses; Names; Pathogenesis; Pathogenicity; Patients; Pattern; Persons; Preparation; Protocols documentation; Rapid diagnostics; Rhizomucor; Ribosomal DNA; Ribosomes; Risk Factors; Running; Sampling; Severities; Specificity; Specimen; Sporothrix; Standardization; Steroids; Technical Degree; Technical Expertise; Technology; Testing; The science of Mycology; Thumb structure; Time; Tornadoes; Trichosporon; Tuberculosis; Virus; Walking; Yeasts; cost; data management; diagnostic assay; diagnostic strategy; emerging pathogen; fungus; health care settings; immunosuppressed; innovation; instrument; molecular diagnostics; molecular sequence database; mortality; nanopore; new technology; novel; novel sequencing technology; novel strategies; pathogenic fungus; portability; programs; rapid test; virtual

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.

抽象的 诊断真菌感染面临着许多挑战，包括所需专业知识的下降 与鉴定真菌相比，减少了专门用于真菌鉴定的仪器和测定的数量 对细菌和病毒。真菌感染患者的病情加剧了这些问题 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，侵袭性感染的分子诊断测定是劳动密集型的，并且需要高 技术专长程度。开发一种有效的检测方法，可以使用技术来替代 古典真菌学专业知识一直很困难。虽然核糖体 ITS 测序是真菌的黄金标准 分子诊断速度慢，需要较高的资金成本，而且对于许多真菌来说，特异性不够。这 该研究将依赖于两种创新成分，这两种成分将能够识别几乎所有真菌 物种等级很快。第一个组成部分是一种称为纳米孔测序的新测序技术， 既快速又便宜。第二个组成部分将包括一个称为 基因间间隔序列（IGS），位于真菌核糖体重复序列内。 IGS 太长，无法 诊断分析中的传统桑格测序可以覆盖，因为从一个序列中生成多个读数 需要引物步行，因此不用于真菌鉴定。然而，纳米孔测序可以 在一次测序运行中轻松覆盖 IGS 区域。达到开发高效锅的目标 真菌诊断策略，三个目标将检验测序很少使用但高度使用的假设 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信息 迄今为止真菌鉴定的特异性和最快的周转时间。这些目标是： 1) 生成 GenBank RefSeq 内的真菌基因间间隔序列参考序列数据库（参考 序列）靶向位点项目，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。长期来看 该项目完成后的目标是未来开发一种快速、廉价、 具有泛真菌能力的测定。