Development of novel field-appropriate differential diagnostics of multiple patho

描述（由申请人提供）：该项目的目标包括开发用于临床和现场条件的新型诊断检测方法。在这个项目中，我们提出了一种新的基于DNA阵列的技术，用于从潜在感染个体中提取的测试样本中进行病原体特异性DNA序列的鲁棒、等温、超灵敏比色检测。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。特异的PNA-DNA结构将成为极具选择性和非常有效的生物标志物。超灵敏度将通过信号扩增的两个步骤来提供：每个rna -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DNAzymes催化多种化学反应，产生化学发光或产生比色输出。这种协同结合了一组经过验证的技术，有望克服长期存在的挑战：在没有靶扩增的情况下检测病原微生物，并将产生一种非常规的、非pcr等温分析，用于权宜之计、万无一失地识别生物防御和新出现的病原体，这是其他技术无法实现的。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理证明数据，这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具体特征（如检测其致病毒力因子，区分耐药菌和药敏菌）大幅扩展我们的病原体鉴定试点数据。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向标准医疗诊断的过渡。一旦为正在研究的病原体开发，联合工作流程将很容易适用于其他临床相关生物体的快速敏感检测。

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.