The development of multiplexed, label-free isothermal diagnostic for rapid identification of bacterial pathogens

 描述（由申请人提供）：快速、灵敏地鉴定临床样本中细菌病原体的能力对于及时、经济有效地启动适当治疗至关重要。考虑到它们克服测定时间和灵敏度限制的固有能力， 方法可以显著影响体外诊断的功效。然而，到目前为止，还没有一种单一的分子方法可以取代或补充临床微生物实验室中的大多数传统培养试验。我们建议通过现有互补技术的协同组合来开发一种集成的体外分子诊断检测方法，以挑战当前基于核酸的诊断范式。我们的方法使用三种工具：先进的肽核酸（PNA）技术，用于高度特异性和选择性的dsDNA序列靶向，使用滚环机制（RCA）确保高灵敏度的序列扩增，以及无标记的微阵列检测技术，用于直接在玻璃表面上扩增的DNA纳米颗粒的快速成像，用于筛选应用。肽核酸技术提供了在dsDNA中产生独特的病原体特异性PNA-DNA构建体，其允许在等温非变性条件下选择性靶向基因组DNA位点。特别设计的核酸引物与该构建体的杂交允许靶序列的选择性RCA扩增并使非特异性基因组物质的扩增最小化。通过增强RCA直接发生在表面上，我们提出通过使用纳米颗粒成像系统来感测扩增的病原体特异性探针，该系统能够检测跨越大的寡聚阵列的扩增产物。该技术的单粒子成像能力允许非常高的灵敏度，具有大量的多路复用，用于同时筛选许多相互作用。我们认为，这些技术的整合可以提高灵敏度和特异性的一个 病原体筛选测定优于目前可用的分子方法，因为序列靶向和扩增中的冗余和选择性步骤。此外，由于该过程的所有步骤都可以在等温条件下进行，因此可以简化或消除热循环所需的仪器。我们提出了一种有望克服长期挑战的策略：无需耗时的培养扩增即可灵敏地检测病原微生物。开发的技术将产生一种非常规的非PCR检测方法，用于快速鉴定其他技术无法实现的细菌和病毒感染。申请的资金将推动这项集成技术向临床相关应用发展，并推动从敏感的研究工具向医疗诊断技术的转变。

 DESCRIPTION (provided by applicant): The ability to rapidly and sensitively identify bacterial pathogens in clinical samples is essential to timely and cost-effective initiation of appropriate therapy. Given their inherent ability to overcome assay time and sensitivity limitations, molecular methods can significantly affect the efficacy of in vitro diagnostics. To date however, no single molecular approach has replaced or supplemented the majority of traditional culture tests in clinical microbiology laboratories. We propose the development of an integrated in vitro molecular diagnostic assay through the synergistic combination of existing complementary techniques to challenge the current nucleic acid-based diagnostic paradigm. Our approach uses three tools: advanced peptide nucleic acid-based (PNA) technology for highly specific and selective dsDNA sequence targeting, sequence amplification using a rolling circle mechanism (RCA) ensuring high sensitivity, and a label-free microarray-amenable detection technique for the rapid imaging of DNA nanoparticles amplified directly on a glass surface for screening applications. Peptide nucleic acid technology affords the creation of unique, pathogen-specific PNA-DNA constructs in dsDNA, which allows selective targeting of genomic DNA sites under isothermal non-denaturing conditions. The hybridization of specially designed nucleic acid primers to this construct allows for selective RCA amplification of the target sequence and minimizing of amplification of non-specific genomic material. By augmenting RCA to occur directly on-surface we propose sensing of the amplified pathogen-specific probes through the use of a nanoparticle imaging system, capable of detecting amplified products across a large oligomeric array. The single particle imaging capability of this technique allows very high sensitivity with substantial multiplexing for screening numerous interactions simultaneously. We believe that the integration of these technologies can improve the sensitivity and specificity of a pathogen screening assay over currently available molecular approaches because of redundant and selective steps in sequence targeting and amplification. Moreover, because all steps of the procedure can be performed under isothermal conditions the instrumentation necessary for thermocycling can be simplified or eliminated. We propose a strategy that is expected to overcome a long-standing challenge: the sensitive detection of pathogenic microorganisms without time-consuming culture amplification. The developed technology will yield an unconventional, non-PCR assay for expedient identification of bacterial and viral infections not achievable by alternative techniques. The requested funding would drive this integrated technology toward clinically relevant application and advance the transition from a sensitive research tool to a medical diagnostic technique.