Isothermal Colorimetric Pathogen DNA Diagnostics

描述（由申请人提供）：本提案的目的是开发一种快速、灵敏的 DMA 诊断测定法，该测定法将新型等温 DNA 扩增反应 (EXPAR) 与通过 DNA 功能化金纳米球进行比色检测相结合，采用适用于护理点设置的格式。 EXPAR 反应可在 55°C 下在几分钟内将短触发寡核苷酸扩增 >10A6 倍。通过 DNA 功能化金纳米球的聚集进行检测可提供红色或蓝色斑点形式的视觉读数。我们将开发一种检测方法来检测 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低下的个体危及生命的感染。 HSV 的广泛存在和耐药菌株的出现需要开发诊断工具，以促进对患者的有效治疗并遏制其进一步传播。长期目标是将这种检测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生成触发寡核苷酸的方法，这将能够特异性识别HSV1和/或HSV2，以及识别耐药突变。 (2) 优化触发寡核苷酸的扩增，并通过 DNA 纳米球聚集进行检测，以单一或多重形式，具有临床应用所需的适当灵敏度、选择性和稳健性。 (3) 优化和验证将触发生成与触发放大和检测相结合的完整测定，以实现对 HSV1、HSV2 及其耐药突变的快速、灵敏、特异、稳健和可重复的检测。该提案的重点是充分开发和优化这种新颖的测定方法，我们计划在后续工作中在微流体装置中实施该测定方法。拟议的项目将有助于在护理点开发基于 DNA 的分子诊断新系统，预计将有助于快速识别和遏制传染源，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.