Development of novel field-appropriate differential diagnostics of multiple patho

描述(由申请人提供)：该项目的目标在于开发用于临床和现场条件的新型诊断分析方法。在这个项目中，我们提出了一种基于DNA阵列的新技术，用于对来自潜在感染者的测试样本中的病原体特定DNA序列进行稳健、恒温、超灵敏的比色检测。一种新的策略是基于肽核酸(PNA)打开独特的短序列(20-30bp)的能力，这使得在非变性条件下检测基因组DNA中的DNA特征序列成为可能。特定的PNA-DNA结构将作为高度选择性和非常有效的生物标记物。超灵敏的信号放大将由两个步骤提供：每个PNA-DNA构建触发聚合酶介导的滚环扩增(RCA)，以产生DNA酶传感器的线性阵列；该DNAzyme催化多次循环的化学反应，产生化学发光或产生比色输出。这种协同结合一套经过验证的技术有望克服长期存在的挑战：在不进行靶标扩增的情况下检测病原微生物，并将产生一种非常规、非PCR等温分析，用于方便、万无一失地鉴定生物防御和替代技术无法实现的新兴病原体。清晰的视觉读数和等温性质使该分析非常适合在资源有限的环境中应用。该项目依赖于我们的原则证明数据，这些数据表明我们能够在模型细菌基因组中检测到大约20个bp长的单拷贝DNA签名位点。在项目期间，我们计划根据基因组的特定特征大幅扩展我们的病原体鉴定试点数据(例如，检测它们的致病毒力因子，区分耐药细菌和敏感细菌)。目前申请的资金将推动技术应用于临床相关病例，并迅速推进从敏感研究工具到标准医疗诊断的过渡。一旦为正在研究的病原体开发，组合的工作流程将很容易适应于对其他临床相关生物体的快速敏感检测。 与公共卫生相关：该项目的目标是开发一种快速、极具特异性和非常敏感的诊断分析方法，用于检测临床样本中出现的病原体，并通过颜色变化反应识别其毒力因素。

DESCRIPTION (provided by applicant): The project's goal consists in the development of novel diagnostic assay for use in clinical and field conditions. In this project we suggest a new DNA array-based technology for robust, isothermal, ultrasensitive colorimetric detection of pathogen-specific DNA sequences in test samples taken from potentially infected individuals. A novel strategy is based on the ability of peptide nucleic acid (PNA) to open up unique short sequences (20- 30bp), which makes it possible to detect DNA signature sequences within genomic DNA under non-denaturing conditions. Specific PNA-DNA construct will serve as exceedingly selective and very effective biomarkers. Ultra-sensitivity will be provided by two steps of signal amplification: each PNA-DNA construct triggers a polymerase-mediated rolling-circle amplification (RCA) to produce a linear array of DNA enzyme sensors; this DNAzymes catalyses chemical reactions with multiple turnover that generate chemiluminescence or produce colorimetric output. This synergistic combination a set of proven techniques is expected to overcome a long standing challenge: the detection of pathogenic microorganisms without target amplification and will yield an unconventional, non-PCR isothermal assay for expedient foolproof identification of biodefense and emerging pathogens not achievable by alternative techniques. The clear visual readouts and isothermal nature make this assay extremely suitable for application in resource-limited setting. The project relies on our proof-of- principle data, which demonstrates that we were able to detect about 20 bp-long, single-copy DNA signature sites in model bacterial genomes. During the project time, we plan to significantly extend our pilot data for pathogen identification based on the specific features of the genome (e.g. detect their pathogenic virulence factors and distinguish drug-resistant and drug-sensitive bacteria). The current requested funding will move technology toward applying it to clinical relevant cases and quickly advance the transition from a sensitive research tool to standard medical diagnostics. Once developed for pathogens under study, the combined workflow would be readily adaptable for rapid sensitive detection of other clinically relevant organisms. PUBLIC HEALTH RELEVANCE: The project's goal consists in the development of a rapid, exceedingly specific and very sensitive diagnostic assay for detection of emerging pathogens in clinical samples and identification of its virulence factors through a color change reaction.