Rapid Point-of-Care Molecular Test for the Differentiation of Dengue and Chikungunya Viruses

用于区分登革热和基孔肯雅病毒的快速护理点分子检测

• 批准号: 8833447
• 负责人: William Nelson
• 金额: $ 16.94万
• 依托单位: TETRACORE, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8833447
• 关键词: Antibodies; Area; Biological Assay; Blood; Blood Glucose; Blood specimen; Centrifugation; Chikungunya virus; Clinical; Clinical Management; Collection; Complex; Country; Coupled; Culicidae; Data; Dengue; Dengue Virus; Detection; Development; Device Designs; Devices; Diagnostic; Ensure; Fever; Fingers; Future; Generations; Home environment; Hospitals; Human; Individual; Infection; Laboratories; Lateral; Liquid substance; Medical; Medical Research; Methods; Molecular; Monitor; Nucleic Acids; Outcome; Patients; Phase; Physicians' Offices; Point-of-Care Systems; Polymerase Chain Reaction; Preparation; Process; Production; Public Health; RNA-Directed DNA Polymerase; Reagent; Resources; Reverse Transcriptase Polymerase Chain Reaction; Running; Sales; Sampling; Serological; System; Temperature; Testing; Time; Touch sensation; Travel; Urbanization; Validation; Viral Load result; Virus; Virus Diseases; Whole Blood; accurate diagnosis; base; chikungunya; climate change; cold temperature; experience; internal control; point of care; point-of-care diagnostics; portability; public health relevance; research clinical testing; response; viral RNA; viral detection

 DESCRIPTION (provided by applicant): Increased human travel, urbanization and climate changes have resulted in a wide geographic spread of febrile illness caused by both Dengue Virus (DENV) and Chikungunya Virus (CHIKV). These two infections show a substantial overlap in clinical presentation. In addition, both are also co-prevalent in many countries. This poses a significant challenge to clinicians, as the two illnesses require different clinical management. A recent infection can only be confirmed by detection of the virus in a blood sample, as positive results in serological assays may instead be indicative of prior infection. However, as antibodies develop in response to the infection, the viral load in the blood decreases. Thus, sensitive and specific detection of these viruses is imperative for an accurate diagnosis. Typically, molecular methods that employ reverse transcriptase real-time polymerase chain reaction (rtRT-PCR) based detection of viral RNA require nucleic acid extraction of whole blood samples. In this Phase I plan, we propose to (1) develop a multiplexed molecular assay for the direct detection of either DENV or CHIKV in blood samples, and then to (2) incorporate this multiplexed assay into a Collect-to-Test (C2T) system in order to overcome the need for complex sample processing. The C2T system includes a simple blood collection and processing method that requires no pipetting or sample manipulation, combined with a cartridge containing the dried-down assay reagents for simultaneous sample preparation and template amplification. The sample preparation and cartridge loading are equivalent to methods currently used to run lateral flow devices. This system will allow for simultaneous detection of DENV and CHIKV by rtRT-PCR from a minimal volume of whole blood (i.e., a finger prick) without the need for centrifugation or time- consuming extraction methods. Critical to this is the development of a rtRT-PCR-based assay optimized for the detection of viral RNA directly from blood samples and requiring no further sample extraction. These reagents, together with the C2T system, will be compatible with the T- COR" 8 portable thermocycler. Tetracore has extensive experience in the development of highly sensitive and specific dried-down, room temperature-stable assays for real-time PCR, including a DENV-specific assay previously developed with Naval Medical Research Center. This assay was systematically evaluated and successfully tested using clinical samples on a commercial laboratory rtRT-PCR platform. Based on our preliminary data and experience we are confident to propose this plan to develop a multiplex rtRT-PCR assay for differential detection of DENV and CHIKV that can be combined for use with the C2T cartridge and the T-COR 8 portable thermocycler, to create an integrated point-of-care system that can be equally effectively in both public health labs and low resource settings.

 描述（由申请人提供）：人类旅行的增加，城市化和气候变化导致登革热病毒（DENV）和基孔肯雅病毒（CHIKV）引起的发热性疾病的广泛地理传播。这两种感染在临床表现上有很大的重叠。此外，这两种疾病在许多国家也同时流行。这对临床医生提出了重大挑战，因为这两种疾病需要不同的临床管理。最近的感染只能通过检测血液样本中的病毒来确认，因为血清学检测的阳性结果可能表明先前的感染。然而，随着抗体对感染的反应，血液中的病毒载量减少。因此，这些病毒的敏感和特异性检测对于准确诊断至关重要。通常，采用基于逆转录酶实时聚合酶链反应（rtRT-PCR）的病毒RNA检测的分子方法需要全血样品的核酸提取。在该I期计划中，我们建议（1）开发用于直接检测血液样品中的DENV或CHIKV的多重分子测定，然后（2）将该多重测定并入收集到测试（C2 T）系统中，以克服对复杂样品处理的需求。C2 T系统包括一种简单的血液采集和处理方法，不需要移液或样品操作，结合含有干燥检测试剂的检测盒，可同时进行样品制备和模板扩增。样品制备和测试卡片加载与目前用于运行侧流装置的方法等同。该系统将允许通过rtRT-PCR从最小体积的全血（即，手指刺破）而不需要离心或耗时的提取方法。这一点的关键是开发一种基于rtRT-PCR的检测方法，该方法可直接从血液样本中检测病毒RNA，无需进一步提取样本。这些试剂连同C2 T系统将与T-COR”8便携式热循环仪兼容。Tetracore在开发用于实时PCR的高灵敏度和特异性干燥、室温稳定的检测方法方面拥有丰富的经验，包括之前与海军医学研究中心开发的DENV特异性检测方法。在商业实验室rtRT-PCR平台上使用临床样本对该检测方法进行了系统评价和成功检测。基于我们的初步数据和经验，我们有信心提出这一计划，以开发一种用于DENV和CHIKV差异检测的多重rtRT-PCR检测方法，该方法可与C2 T检测盒和T-COR 8便携式热循环仪结合使用，以创建一种在公共卫生实验室和低资源环境中同样有效的综合床旁系统。