A Sensitive, Quantitative, Low-cost, Fluorescence Diagnostic System for Dengue

一种灵敏、定量、低成本的登革热荧光诊断系统

• 批准号: 8780736
• 负责人: Linda G Lee
• 金额: $ 13.47万
• 依托单位: SONG DIAGNOSTIC RESEARCH, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8780736
• 关键词: Acute; Antibodies; Antigen-Antibody Complex; Area; Bedside Testings; Binding; Biological Assay; Cessation of life; Clinical; Clinical Trials; Complex; Computer software; Coupled; Culicidae; Data; Data Analyses; Dengue; Dengue Virus; Detection; Devices; Diagnosis; Diagnostic; Disease; Dissociation; Dyes; Enrollment; Enzyme-Linked Immunosorbent Assay; Epidemiology; Eye; Fever; Fluorescence; Fluorescent Dyes; Glosso-Sterandryl; Glycoproteins; Goals; Gold; Healthcare; Hemorrhagic Shock; Human; Human Chorionic Gonadotropin; Illness Days; Image Analysis; Immunoassay; Immunoglobulin G; Immunoglobulin M; Improve Access; Individual; Infection; Investigation; Laboratories; Lateral; Lighting; Malaria; Molecular; Nicaragua; Optics; Outcome; PTPN11 gene; Patients; Performance; Phase; Phycoerythrin; Process; Property; Proteins; Reagent; Reporter; Reporting; Resources; Reverse Transcription; Sampling; Serotyping; Serum; Signal Transduction; Site; Specificity; Sri Lanka; System; Technology; Telephone; Testing; Time; Universities; Viral; Viral Hemorrhagic Fevers; Virus; Virus Diseases; Work; base; cloud based; cost; design; detector; global health; high risk; improved; internal control; laboratory facility; pathogen; point of care; point-of-care diagnostics; prototype; public health relevance; reagent testing; secondary infection; technology development; vector

DESCRIPTION (provided by applicant): Dengue is the most rapidly spreading mosquito-borne virus in the world and currently infects 390 million people annually. Studies have shown that existing point-of-care tests have inadequate sensitivity to identify many individuals infected with dengue. None have the quantitative capability necessary to aid in the detection of severe dengue, especially in secondary dengue infections. Our approach will provide a low-cost yet quantitative and sensitive system. Our lateral flow immunoassay (LFIA) test will analyze and quantitate the presence of dengue virus non-structural protein (DENV NS1), an analyte that is frequently used to determine a diagnosis of dengue. Fluorescent dyes will be used to improve the limit of detection compared to existing rapid tests. A smart phone is utilized for imaging, analysis and automated reporting; the latter feature will simplify disease tracking. A long Stokes shift dye, R-phycoerythrin (PE) will be used as the fluorescent reporter, either singly or bundled into a cluster. The optical properties of PE allow the use of an inexpensive illumination device; the minimal non-specific binding of PE results in sensitive detection with a wide dynamic range. Additional gains in sensitivity will be achieved through an integrated test feature designed to dissociate immune complexed protein for improved access by the test reagents. In Phase I we will: 1) Optimize the PE reporter (cluster) on anti-NS1 antibody; 2) Develop a smartphone application to allow automated analysis; 3) Develop an on-LFIA strip "zone of dissociation" to aid in analysis of complexed NS1; 4) Develop the use of the control line on the lateral flow strip to allow absolute quantitation of NS1; 5) Design, fabricate and test a prototype of a low cost fluorescence system designed with a materials cost less than $35. 6) Validate the NS1 LFIAs by comparing results with commercially available ELISA and LFIA tests using patient samples. These investigations will demonstrate the performance advantage of our system for detection and analysis of NS1. For phase II we hope to include multiple analytes, such as DENV IgG and IgM, or a multiplex febrile disease test strip (e.g. malaria and DENV).

描述(申请人提供)：登革热是世界上传播最快的蚊媒病毒，目前每年感染3.9亿人。研究表明，现有的医疗点检测不足以识别许多感染了 登革热。它们都没有必要的定量能力来帮助检测严重登革热，特别是在继发性登革热感染中。我们的方法将提供一个低成本但定量和敏感的系统。我们的横向流动免疫分析(LFIA)测试将分析和定量登革病毒非结构蛋白(DENV NS1)的存在，这是一种经常用于确定登革热诊断的分析物。与现有的快速检测相比，将使用荧光染料来提高检测极限。智能手机用于成像、分析和自动报告；后一种功能将简化疾病跟踪。一种长斯托克斯位移染料，R-藻红蛋白(PE)将被用作荧光报告，可以单独使用，也可以捆绑成簇。PE的光学特性允许使用廉价的照明设备；PE的最小非特异性结合导致具有宽动态范围的灵敏检测。通过设计一种旨在分离免疫复杂蛋白的集成测试功能，将进一步提高灵敏度，以改善测试试剂的访问。在第一阶段，我们将：1)优化抗NS1抗体的PE报告器(簇)；2)开发智能手机应用程序，以实现自动化分析；3)开发On-LFIA条带“解离区”，以帮助分析复杂的NS1；4)开发横向流动条上的控制线，以实现NS1的绝对定量；5)设计、制造和测试低成本荧光系统的原型，设计材料成本低于35美元。6)通过将结果与商业上可用的ELISA法和使用患者样本的LFIA检测结果进行比较，验证NS1 LFIA。这些研究将展示我们的系统在检测和分析NS1方面的性能优势。对于第二阶段，我们希望包括多种分析，如DENV免疫球蛋白和IgM，或多重热病试纸(例如疟疾和DENV)。