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诊断检测方法，该方法将新型等温DNA扩增反应（EXPAR）与通过DNA功能化金纳米球进行的比色检测相结合，适用于即时护理环境。EXPAR反应在55 ° C下在几分钟内扩增短触发寡核苷酸> 10 ~ 6倍。通过DNA官能化的金纳米球的聚集的检测提供了红色或蓝色斑点形式的视觉读数。我们将开发检测1型和2型单纯疱疹病毒（HSV 1和HSV 2）的检测方法，虽然通常是良性的，但可导致新生儿和免疫功能低下个体的危及生命的感染。HSV的广泛发生和耐药菌株的出现使得有必要开发诊断工具，以促进对患者的有效治疗并遏制进一步传播。长期目标将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产生触发寡核苷酸的方法，这将能够特异性鉴定HSV 1和/或HSV 2，以及鉴定耐药突变。(2)优化通过DNA纳米球聚集与检测偶联的触发寡核苷酸的扩增，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)优化并验证将联合收割机触发物生成与触发物扩增和检测相结合的完整检测方法，以实现对HSV 1、HSV 2及其耐药突变的快速、灵敏、特异、稳健和可重现检测。该提案的重点是充分开发和优化这种新的检测方法，我们计划在随后的努力中在微流体设备中实施。拟议的项目将能够在护理点环境中开发基于DNA的分子诊断新系统，预计这将有助于快速识别和遏制传染性病原体，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.