Isothermal Colorimetric Pathogen DNA Diagnostics

描述(申请人提供)：这项建议的目标是开发一种快速、灵敏的DMA诊断分析，它结合了一种新颖的等温DNA扩增反应(EXPAR)与通过DNA功能化的金纳米球进行比色检测的适用于护理场所的格式。EXPAR反应在55℃下扩增短触发寡核苷酸&10A6倍，大约在几分钟内。通过聚集DNA功能化的金纳米球进行检测，可以提供红色或蓝色斑点形式的可视读数。我们将开发这种检测方法来检测1型和2型单纯疱疹病毒(HSV1和HSV2)，这种病毒虽然通常是良性的，但可能会导致新生儿和免疫功能受损的人发生危及生命的感染。单纯疱疹病毒的广泛发生和耐药株的出现要求开发诊断工具，以促进对患者的有效治疗并遏制进一步的传播。长期目标将是将这种分析形式扩展到其他类别的病原体。该项目的具体目标是：(1)开发从HSV基因组靶DNA中产生触发寡核苷酸的方法，这将使HSV1和/或HSV2的特异性识别以及耐药突变的识别成为可能。(2)通过DNA纳米球聚合，以单一或多重形式优化触发寡核苷酸的扩增，使其具有临床应用所需的适当的灵敏度、选择性和稳健性。(3)优化和验证将触发产生与触发扩增和检测相结合的完整检测方法，以实现对HSV1、HSV2及其耐药性突变的快速、灵敏、特异、可靠和可重复性的检测。这项建议的重点是充分开发和优化这一新的检测方法，我们计划在后续的工作中在微流控设备中实施。拟议的项目将能够在护理场所开发新的基于DNA的分子诊断系统，预计这将有助于快速识别和遏制传染病病原体，从而对人类健康产生重大影响。

DESCRIPTION (provided by applicant): The objective of this proposal is to develop a rapid, sensitive DMA diagnostic assay, which combines a novel isothermal DNA amplification reaction (EXPAR) with colorimetric detection through DNA-functionalized gold nanospheres in a format applicable to point-of-care settings. The EXPAR reaction amplifies short trigger oligonucleotides >10A6 fold at 55¿C in a matter of minutes. Detection through the aggregation of DNA- functionalized gold nanospheres provides a visual readout in the form of a red or blue spot. We will develop the assay to detect Herpes Simplex Virus type 1 and 2 (HSV1 and HSV2), which, although generally benign, can lead to life-threatening infections in newborns and immuno-compromised individuals. The widespread occurrence of HSV and the emergence of drug-resistant strains necessitate the development of diagnostic tools to facilitate effective treatment of patients and to contain further spread. The long-term goal will be to expand this assay format to other classes of pathogens. The specific aims of the proposed project are to: (1) Develop methods for generating trigger oligonucleotides from HSV genomic target DNA, which will enable specific identification of HSV1 and/or HSV2, as well as identification of drug-resistance mutations. (2) Optimize the amplification of trigger oligonucleotides coupled to detection via DNA nanosphere aggregation, in single or multiplexed format, with the appropriate sensitivity, selectivity and robustness required for clinical applications. (3) Optimize and validate complete assays that combine trigger generation with trigger amplification and detection to enable rapid, sensitive, specific, robust, and reproducible detection of HSV1, HSV2, and drug resistance mutations thereof. This proposal focuses on fully developing and optimizing this novel assay, which we plan to implement in a micro fluidic device in a subsequent effort. The proposed project will enable the development of new systems for DNA-based molecular diagnostics in point of care settings, which is expected to facilitate rapid identification and containment of infectious agents, and thus have a significant impact on human health.