Isothermal Colorimetric Pathogen DNA Diagnostics

等温比色病原体 DNA 诊断

• 批准号: 7069468
• 负责人: Angelika Niemz
• 金额: $ 18.59万
• 依托单位: KECK GRADUATE INST OF APPLIED LIFE SCIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7069468
• 关键词: DNA; drug resistance; gene mutation; oligonucleotides

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.

描述（由适用提供）：该提案的目的是开发快速，敏感的DMA诊断测定法，该测定方法结合了一种新型的等温DNA扩增反应（Expar）和通过DNA官能化的金纳米圈的比色检测，适用于适用于点的护理设置的形式。伸出反应放大器在几分钟内以55 c触发寡核苷酸> 10a6倍。通过DNA官能化的金纳米球的聚集来检测，以红色或蓝色点的形式提供了视觉读数。我们将开发用于检测1型和2型单纯疱疹病毒（HSV1和HSV2）的测定法，尽管通常良性可以导致新生儿和免疫受损的个体威胁生命的感染。 HSV发生的宽度和抗药性菌株的出现需要开发诊断工具，以促进有效治疗患者并遏制进一步扩散。长期目标是将这种测定格式扩展到其他类别的病原体。拟议项目的具体目的是：（1）开发从HSV基因组靶DNA产生触发寡核苷酸的方法，该靶向DNA将对HSV1和/或HSV2进行特定鉴定以及药物抗性突变的鉴定。 （2）优化以单个或多重格式的DNA纳米圈聚集的触发寡核苷酸的扩增，具有适当的灵敏度，选择性和鲁棒性。 （3）优化和验证完整的测定，将触发器的产生与触发扩增和检测相结合，以实现HSV1，HSV2和其耐药性突变的快速，敏感，特定，健壮和可再现的检测。专注于充分开发和优化这种新颖的测定法，我们计划在随后的努力中在微流体设备中实施。拟议的项目将在护理环境中为基于DNA的分子诊断的新系统开发，这有望促进快速识别和遏制感染剂，从而对人类健康产生重大影响。