Isothermal Colorimetric Pathogen DNA Diagnostics

描述（由申请人提供）：本提案的目标是开发一种快速、敏感的DMA诊断检测方法，该方法结合了一种新型的等温DNA扩增反应（EXPAR）和通过DNA功能化金纳米球进行比色检测的方法，其格式适用于护理点设置。EXPAR反应在55℃下在几分钟内扩增短触发寡核苷酸。通过DNA功能化金纳米球的聚集检测，以红色或蓝色点的形式提供视觉读数。我们将开发检测1型和2型单纯疱疹病毒（HSV1和HSV2）的检测方法，这两种病毒虽然通常是良性的，但在新生儿和免疫功能低下的个体中可能导致危及生命的感染。单纯疱疹病毒的广泛发生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进一步传播。长期目标将是将这种分析格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中生成触发寡核苷酸的方法，这将使HSV1和/或HSV2的特异性鉴定以及耐药性突变的鉴定成为可能。(2)优化触发寡核苷酸通过DNA纳米球聚集偶联检测的扩增，以单或多路形式，具有临床应用所需的适当灵敏度、选择性和鲁棒性。(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HSV1、HSV2及其耐药突变的快速、敏感、特异性、鲁棒性和可重复性检测。本提案的重点是充分开发和优化这种新的分析方法，我们计划在随后的努力中在微流体装置中实施。拟议的项目将使在护理点环境中开发基于dna的分子诊断新系统成为可能，预计这将有助于迅速查明和控制传染因子，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.