A Sensitive and Serotype-Specific Dengue Diagnostic Test for Low-Resource Setting

适用于资源匮乏环境的敏感且血清型特异性的登革热诊断测试

• 批准号: 8713439
• 负责人: SEASON S-S WONG
• 金额: $ 19.99万
• 依托单位: AI BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8713439
• 关键词: Acute; Aedes; Affect; Antibodies; Antigens; Area; Biological Assay; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Characteristics; Climate; Clinical; Clinical Management; Copper; Country; Culicidae; Dengue; Dengue Hemorrhagic Fever; Dengue Virus; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Early Diagnosis; Environment; Evaluation; FDA approved; Fever; Health; Infection; Influenza; Laboratories; Laboratory Diagnosis; Lead; Leptospirosis; Life; Magnetism; Malaria; Medical; Methods; Molecular; Morbidity - disease rate; Nucleic Acids; Outcome; Patients; Performance; Phase; Plasma; Polymerase Chain Reaction; Population; Populations at Risk; Preparation; Process; Public Health; Puerto Rico; RNA; RNA Viruses; Reaction; Readiness; Reagent; Resources; Reverse Transcription; Risk; Running; Sampling; Scheme; Sensitivity and Specificity; Serotyping; Serum; Signs and Symptoms; Small Business Innovation Research Grant; Social Welfare; Speed; Surveys; Symptoms; Syringes; System; Technology; Temperature; Testing; Time; Training; United States Virgin Islands; Vaccines; Viral; Viral Load result; Virus; Virus Diseases; Whole Blood; Work; base; cost; design; detector; improved; innovation; instrument; meetings; mortality; particle; point of care; prevent; public health relevance; transmission process; viral RNA

DESCRIPTION (provided by applicant): Dengue is transmitted mainly by the Aedes aegypti mosquitoes which inhabit the tropics, making Dengue endemic to these areas. It is caused by four genetically and serologically related viruses, termed DENV1, DENV2, DENV3, and DENV4. Dengue infection is a leading cause of illness and death in the tropics and subtropics, including Puerto Rico and the U.S. Virgin Islands, where thousands of U.S. citizens develop dengue fever every year. With three billion of the world's population at risk from dengue, an estimated 50-100 million cases of Dengue Fever (DF), and hundreds of thousands of cases of severe dengue (previously known as Dengue Hemorrhagic Fever or DHF) that occur every year, the demand for a rapid, sensitive, and serotype- specific dengue diagnostics test is high. Patients would be diagnosed sooner to receive treatment, and public health laboratories would have a clearer picture of the true number of dengue cases. Dengue is an acute illness where most patients are present with symptoms when they are viremic, but have not yet developed antibodies. Therefore, the early detection of viral components such as RNA or antigens is critical. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays targeting the dengue virus in whole blood or serum have been demonstrated to provide serotype-specific and early diagnostics, but it also requires sophisticated instruments to perform the test. In this SBIR Phase I application, we propose to develop a highly specific and sensitive system to detect the presence of dengue viral RNA. Using our self-contained sample preparation cartridge (SPC) technology and a highly robust convective PCR thermal cycler, we will build a low- cost, multi-sample, and integrated system that takes full advantage of a CDC developed, FDA-approved serotype specific real-time RT-PCR dengue diagnostic assay. Nucleic acid extraction in low-resource settings (LRS) for molecular diagnostics is difficult to perform, but through the use of our self-contained SPC, NA extraction can be done in a safe and enclosed environment in less than 10 min. The extracted NA (RNA in this case) can be ejected from the SPC and used for real-time RT-PCR reaction using a rapid and robust convective PCR thermal cycler. Convective PCR is significantly simpler to operate than traditional PCR while maintaining the specificity, sensitivity, and multiplex capability of PCR. The speed of cPCR is also comparable to isothermal amplification. Using our system, we will enable identification of the serotype and quantitative determination of the viral load in less than 30 min of RT-PCR reaction. Unlike other "single-sample" integrated systems that are being developed, our approach allows multi-sample processing and molecular detection without significantly adding cost to the system. This means our approach will lead to an assay that can be practically and cost-effectively implemented in LRS. We will work closely with the CDC Dengue Branch in Puerto Rico to integrate the serotype-specific dengue RT-PCR assay into our system to deliver an effective dengue assay without compromised performance. At the conclusion of the Phase I, we will demonstrate the technology to our collaborator at CDC's Dengue Branch.

描述(申请人提供)：登革热主要由生活在热带地区的埃及伊蚊传播，使登革热成为这些地区的地方性疾病。它由四种基因和血清学相关的病毒引起，分别称为DENV1、DENV2、DENV3和DENV4。登革热感染是热带和亚热带地区疾病和死亡的主要原因，包括波多黎各和美属维尔京群岛，那里每年有数千名美国公民患上登革热。世界上有30亿人口面临登革热的风险，估计有5000-1亿登革热(DF)病例，以及每年发生的数十万严重登革热(以前称为登革出血热或DHF)，因此对快速、敏感和具有血清型特异性的登革热诊断测试的需求很高。患者将得到更早的诊断以接受治疗，公共卫生实验室将更清楚地了解登革热病例的真实数量。登革热是一种急性疾病，大多数患者在感染病毒时出现症状，但尚未产生抗体。因此，对RNA或抗原等病毒成分的早期检测至关重要。针对全血或血清中登革病毒的实时逆转录聚合酶链式反应(RT-PCR)分析已被证明可以提供特定的血清型和早期诊断，但它也需要复杂的仪器来执行测试。在这个SBIR第一阶段的应用中，我们建议开发一种高度特异和敏感的系统来检测登革热病毒RNA的存在。使用我们独立的样品制备盒(SPC)技术和高度坚固的对流PCR热循环仪，我们将建立一个低成本、多样品和集成的系统，该系统充分利用美国疾病控制与预防中心开发的FDA批准的特定血清型实时RT-PCR登革热诊断分析。用于分子诊断的低资源环境(LRS)中的核酸提取很难执行，但通过使用我们独立的SPC，NA提取可以在安全和封闭的环境中在不到10分钟内完成。提取的NA(在这种情况下是RNA)可以从SPC中喷射出来，并使用快速而坚固的对流PCR热循环器用于实时RT-PCR反应。与传统的聚合酶链式反应相比，对流聚合酶链式反应操作简单，同时保持了聚合酶链式反应的特异性、敏感性和多重能力。CPCR的速度也与等温扩增相当。使用我们的系统，我们将能够在不到30分钟的RT-PCR反应中鉴定血清型和定量测定病毒载量。与其他正在开发的“单样品”集成系统不同，我们的方法允许进行多样品处理和分子检测，而不会显著增加系统的成本。这意味着我们的方法将导致一种可以在LRS中实际和经济有效地实施的分析。我们将与波多黎各疾病控制与预防中心登革热分支机构密切合作，将血清特异性登革热RT-PCR检测整合到我们的系统中，在不影响性能的情况下提供有效的登革热检测。在第一阶段结束时，我们将向疾控中心登革热分部的合作者展示这项技术。

项目成果

Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care.

• DOI: 10.1021/acs.analchem.5b04153
• 发表时间: 2016-05-03
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Priye A;Wong S;Bi Y;Carpio M;Chang J;Coen M;Cope D;Harris J;Johnson J;Keller A;Lim R;Lu S;Millard A;Pangelinan A;Patel N;Smith L;Chan K;Ugaz VM
• 通讯作者: Ugaz VM