Barcoded Nanowires for Multiplexed Clinical Diagnostics

用于多重临床诊断的条形码纳米线

• 批准号: 7096320
• 负责人: CHRISTINE D KEATING
• 金额: $ 29.65万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-04 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7096320
• 关键词: culture; diagnosis; fluorescence; metals; motivation; nucleic acid quantitation /detection; particle; respiratory infections; respiratory virus; tissue /cell culture

DESCRIPTION (provided by applicant): We propose to develop rapid multiplexed assays for the diagnosis of respiratory pathogens in clinical samples. Many viral and bacterial infections cause respiratory illnesses with very similar symptoms, thus, timely and accurate pathogen identification is important for patient recovery and public health monitoring. Current methods include direct immunofluorescence and cell culture, both of which currently require individual tests for each pathogen. We will use bar-coded metallic nanowires as encoded supports for two types of multiplexed diagnostic assays, which will be tested on clinical samples and ultimately transitioned into a clinical setting during the course of the proposed work. Aim 1. Self quenching nanowire beacons. We will take advantage of the electromagnetic effects near metal surfaces (quenching, enhancement, and alteration of fluorescence lifetimes), to produce nanowire beacon probes that enable many assays to be conducted simultaneously in the same sample. The resulting multiplexible, "closed tube" nucleic acid detection assays will require no target labeling or washing, nor any special instrumentation beyond a conventional fluorescence optical microscope, and are therefore amenable to point-of-contact assays such as clinical diagnostics. We propose here to develop a 17-plexed assay for respiratory pathogens. Aim 2. Simultaneous multiplexed amplification and immobilization for pathogen detection. For viruses shed at very low levels, amplification (by growing in culture or by PCR) is necessary for detection. We will overcome current limitations of viral detection by existing methods by introducing multiplexed amplification and immobilization onto bar-coded nanowires (MAIN). To accomplish this we will develop robust surface attachment chemistry that can withstand thermo cycling and will provide for minimal steric hindrance of hybridization and extension, and will then use these advances to simultaneously direct all PCR products of interest onto different, visually identifiable nanowire supports for sensitive pathogen detection.

描述（由申请方提供）：我们建议开发用于诊断临床样本中呼吸道病原体的快速多重检测试剂盒。许多病毒和细菌感染引起的呼吸道疾病具有非常相似的症状，因此，及时准确的病原体识别对于患者康复和公共卫生监测非常重要。目前的方法包括直接免疫荧光和细胞培养，这两种方法目前都需要对每种病原体进行单独检测。我们将使用条形码金属纳米线作为两种类型的多重诊断检测的编码支持，这将在临床样本上进行测试，并最终在拟议的工作过程中过渡到临床环境。目标1.自猝灭纳米线信标。我们将利用金属表面附近的电磁效应（猝灭，增强和荧光寿命的改变），生产纳米线信标探针，使许多测定在同一样品中同时进行。所得的多重“封闭管”核酸检测测定将不需要靶标记或洗涤，也不需要常规荧光光学显微镜之外的任何特殊仪器，并且因此适合于接触点测定，例如临床诊断。我们建议在这里开发一个17重检测呼吸道病原体。目标二。用于病原体检测的同时多重扩增和固定。对于以非常低的水平脱落的病毒，扩增（通过在培养物中生长或通过PCR）对于检测是必要的。我们将通过引入多重扩增和固定到条形码纳米线（MAIN）上来克服现有方法检测病毒的局限性。为了实现这一点，我们将开发强大的表面附着化学，可以承受热循环，并将提供最小的空间位阻的杂交和延伸，然后将使用这些进步，同时引导所有的PCR产物到不同的，视觉上可识别的纳米线支持敏感的病原体检测。