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

描述（由申请人提供）：登革热主要由居住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病。它由四种遗传和血清学相关的病毒引起，称为DENV 1，DENV 2，DENV 3和DENV 4。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，每年有数千名美国公民患上登革热。由于世界上有30亿人口处于登革热的风险中，估计有5000万至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）病例，因此对快速、敏感和血清型特异性登革热诊断测试的需求很高。患者将更快地被诊断出来接受治疗，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，大多数患者在病毒血症时出现症状，但尚未产生抗体。因此，病毒成分如RNA或抗原的早期检测至关重要。针对全血或血清中登革热病毒的实时逆转录聚合酶链反应（RT-PCR）检测已被证明可提供特定类型的早期诊断，但它也需要复杂的仪器来进行测试。在SBIR第一阶段的应用中，我们建议开发一种高度特异性和灵敏度的系统来检测登革病毒RNA的存在。使用我们的独立样品制备盒（SPC）技术和高度稳健的对流PCR热循环仪，我们将构建一种低成本、多样品和集成系统，其充分利用CDC开发的、FDA批准的血清型特异性实时RT-PCR登革热诊断测定。用于分子诊断的低资源环境（LRS）中的核酸提取很难进行，但通过使用我们的独立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（在这种情况下为RNA）可以从SPC中排出，并使用快速和强大的对流PCR热循环仪用于实时RT-PCR反应。与传统PCR相比，对流PCR操作简单，同时保持了PCR的特异性、灵敏度和多重性。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，我们的方法允许多样品处理和分子检测，而不会显著增加系统成本。这意味着我们的方法将导致一种可以在LRS中实际且具有成本效益地实施的测定。我们将与波多黎各的疾病预防控制中心登革热分支密切合作，将特定型登革热RT-PCR检测方法整合到我们的系统中，以提供有效的登革热检测方法，而不会影响性能。在第一阶段结束时，我们将向疾病预防控制中心登革热分支的合作者展示这项技术。

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.