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

 描述(由申请人提供)：快速和灵敏地识别临床样本中的细菌病原体的能力对于及时和具有成本效益的适当治疗的启动至关重要。鉴于它们固有的克服检测时间和灵敏度限制的能力，分子 方法可以显著影响体外诊断的效果。然而，到目前为止，还没有一种单一的分子方法取代或补充临床微生物学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方法，以挑战当前基于核酸的诊断范式。我们的方法使用了三种工具：先进的基于肽核酸的(PNA)技术，用于高度特异性和选择性的dsDNA序列靶向；使用确保高灵敏度的滚圈机制(RCA)的序列扩增；以及无标记微阵列兼容检测技术，用于快速成像直接在玻璃表面扩增的DNA纳米颗粒用于筛选应用。肽核酸技术提供了在dsDNA中创建独特的、病原体特异性的PNA-DNA结构，从而允许在等温非变性条件下选择性地靶向基因组DNA位点。将专门设计的核酸引物与该构建物杂交，可以选择性地对目标序列进行RCA扩增，并将非特异性基因组材料的扩增降至最低。通过将RCA增加到直接发生在表面上，我们建议通过使用纳米颗粒成像系统来检测放大的病原体特异性探针，从而能够检测到大型寡聚阵列中的扩增产物。这项技术的单粒子成像能力允许非常高的灵敏度和大量的多路复用，以同时筛选多种相互作用。我们相信，这些技术的结合可以提高一种 由于序列靶向和扩增中的冗余和选择性步骤，目前可用的分子方法无法进行病原体筛选。此外，由于该程序的所有步骤都可以在等温条件下进行，因此可以简化或取消热循环所需的仪器。我们提出了一种策略，有望克服长期存在的挑战：无需耗时的培养扩增即可灵敏地检测病原微生物。这项开发的技术将产生一种非传统的、非聚合酶链式反应的分析方法，用于方便地识别细菌和病毒感染，这是替代技术无法实现的。申请的资金将推动这项集成技术在临床上的应用，并推动从敏感的研究工具向医学诊断技术的过渡。

 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.