Rapid and Low- cost POC Assay for Zika Virus Diagnosis

项目总结 这项提议的目的是开发一种快速、低成本、易用的寨卡病毒POC诊断测试 (ZIKV)。ZIKV是一种新兴的蚊媒黄病毒，由于新的证据而引起了全球的关注 它与出生缺陷小头畸形有关。此外，健康专家也对这种联系感到担忧 该病毒与格林-巴利综合征(GBS)之间的关系。奥巴马总统于2016年2月初宣布 他正在要求国会提供超过18亿美元的紧急资金，以扩大美国的努力 抗击寨卡病毒。应对寨卡病毒这样的疫情最重要的工具之一是可靠的， 准确、易用的诊断测试。现有测试的问题在于，它们往往无法区分 寨卡病毒与生活在中南美洲的人们经常接触的类似病毒之间的关系。 疾病预防控制中心建议使用分子检测(例如，逆转录聚合酶链法 反应或RT-PCR)以确定疑似病例中的寨卡病毒。而疾控中心和合格的州健康 部门实验室可以进行RT-PCR ZIKV测试，这些实验室使用高度复杂的仪器和 周转时间通常为几周。为此，我们将与国际知名的虫媒病毒合作 德克萨斯大学医学部专家、美国领先的ZIKV研究员Scott Weaver教授 (UTMB)开发一种低成本的POC系统，用于在非传统医疗环境中检测ZIKV感染 美国和美洲。该装置使用上发条的音乐盒来驱动创新设计的塑料齿轮组 在预填充盒中自动提取核酸，并将纯化的RNA直接洗脱成实时 掌握逆转录重组酶聚合酶扩增等扩增方法的组合 反应(RT-RPA)或聚合酶链式反应(RT-PCR)。该齿轮组和一次性墨盒可以 为了生物安全和防止污染，请将其密封。这种便携式系统缺少复杂的微流体， 泵或其他敏感/高成本部件，因此适用于资源有限的国家。这个 简单的样本到答案流程只需要最少的培训，并将患者的 诊断状态。在第一阶段，我们将首先演示从添加了添加剂的血清和尿液中提取高质量的rna。 使用预先装满的墨盒和齿轮组在10分钟内取样。我们还将证明洗脱出的RNA 使用等温实时RT-RPA可以快速扩增样品以确认ZIKV RNA的存在 化验。我们的目标是在15分钟内完成实时RT-RPA扩增反应。《荧光报》 放大产生的信号将使用LED激励和智能手机摄像头进行监控 排放监测和分析。在第一阶段接近尾声时，我们的协作者将为ZIKV验证我们的设备 侦测。我们技术的成功开发和商业化将导致分子 在非实验室环境中实施诊断，并将帮助资源有限的人群获得 检测出寨卡病毒。简而言之，我们的便携式系统将与正在进行的ZIKV全球卫生紧急情况相关。

PROJECT SUMMARY The aim of this proposal is to develop a rapid, low-cost, and easy-to-use POC diagnostic test for Zika virus (ZIKV). ZIKV is an emerging mosquito-borne Flavivirus that has gained global attention due to new evidence that it is linked to birth defect microcephaly. Additionally, health experts are also concerned about the link between the virus and Guillain-Barré syndrome (GBS). President Obama announced in early February of 2016 that he is asking Congress for more than $1.8 billion in emergency funding to expand the United States’ efforts to combat Zika. One of the most vital tools necessary to confront an outbreak like the Zika virus is a reliable, accurate, and easily accessible diagnostic test. The issue with existing tests is that they often fail to distinguish between Zika and similar viruses to which people living in Central and South America are frequently exposed. The CDC has recommended the use of molecular detection (e.g., reverse transcription polymerase chain reaction or RT-PCR) to identify Zika virus in suspected cases. While CDC and qualified state health department labs can perform the RT-PCR ZIKV test, these labs use highly sophisticated instruments and turnaround times are typically weeks. To this end, we will collaborate with internationally known arboviruses expert and leading US ZIKV researcher Professor Scott Weaver at the University of Texas Medical Branch (UTMB) to develop a low-cost POC system for testing ZIKVs infection in non-traditional healthcare settings in the US and Americas. This device uses a wind-up music box to drive an innovatively designed plastic gear set to automate nucleic acid extraction in pre-filled cartridges and to elute the purified RNA directly into real-time master mix of amplification assays such as reverse transcription recombinase polymerase amplification reaction (RT-RPA) or polymerase chain reactions (RT-PCR). The gear set and the disposable cartridge can be enclosed for biosafety and contamination prevention. This portable system lacks complicated microfluidics, pumps or other sensitive/high cost components, thus making it suitable for resource-limited countries. The simple sample-to-answer process requires minimal training and informs healthcare workers of patients’ diagnostic statuses. In Phase I, we will first demonstrate quality RNA extraction from spiked serum and urine samples in 10 min using pre-filled cartridges and the gear set. We will also demonstrate that the eluted RNA samples can be rapidly amplified to confirm the presence of ZIKV RNA using isothermal real-time RT-RPA assay. We aim to complete real-time RT-RPA amplification reactions in less than 15 min. The fluorescent signal produced from amplification will be monitored using LEDs for excitation and a smartphone camera for emission monitoring and analysis. Near the end of Phase I, our collaborator will validate our device for ZIKV detection. The successful development and commercialization of our technology will lead to molecular diagnostics being implemented in non-laboratory settings and will assist resource-limited populations to get tested for Zika. In short, our portable system will be pertinent to the ongoing ZIKV global health emergency.