A Rapid, Low-Cost Point of Care Diagnostic for detection of Zika virus RNA

摘要 寨卡病毒(ZIKV)迅速出现并在中南美洲、加勒比海地区和 波多黎各自2007年密克罗尼西亚最近一次疫情爆发以来。由蚊子伊蚊传播，其预测 传播将对美国东南部产生重大影响。大多数寨卡病毒感染仍无症状或存在 有非特异性皮疹和发热；因此，很难诊断和报告。然而，有两个 主要的健康后果似乎与ZIKV疫情有关，这使它有别于其他 黄病毒，如西尼罗河病毒和登革热；即：(A)从受感染的母亲传播给胎儿 导致胎儿小头畸形的报告；(B)成人的格林-巴利综合征(GBS)。几个 研究小组现在已经开发出检测寨卡病毒的qRT-PCR检测方法。然而，这样的测试相对昂贵， 需要设备齐全的实验室和专门的设备，而且这个过程至少需要3个小时 完成。迫切需要一种快速、灵敏、特异、经济的寨卡病毒诊断试验。 这种化验方法可以常规地用于资源匮乏的环境以及医生的办公室，包括作为 常规的产前护理。因此，SBIR第一阶段项目的目标是使用回路中介的等温 核酸扩增(LAMP)，以开发一种快速、灵敏的寨卡病毒诊断方法。这 技术的复杂性很低，只需要一个水浴。比色结果可用肉眼在 一小时或更少。我们将使用ZIKV和其他病毒(包括西尼罗河病毒、登革热)的连续稀释 病毒和基孔肯雅病毒)在人的血液、唾液和尿液中添加，以确定该检测的灵敏度 和专一性。基于我们实验室在LAMP检测开发方面的丰富经验，我们 预测10-100个病毒基因组的检测下限，具有非常高的特异性。除了快速， 在比色法ZIKV检测中，我们还将开发一种快速、侧向流动分析方法，以方便差示分析 与其他黄病毒一起传播。快速护理点检测的发展将减少对中枢神经系统的依赖 用于流行病学监测和临床诊断的实验室检测设施，是 目前疫情流行的资源贫乏地区。此外，我们预计可供选择的 快速检测将导致将ZIKV检测纳入对以下妇女的可持续的常规评估 怀孕或预期怀孕，以及他们的伴侣。

ABSTRACT Zika virus (ZIKV) has rapidly emerged and spread through South and Central America, the Caribbean, and Puerto Rico since its last outbreak in Micronesia in 2007. Transmitted by the mosquito Aedes sp, its forecasted spread will have a major impact on the Southeast U.S. Most ZIKV infections remain asymptomatic or present with non-specific rash and fever; therefore, they have been difficult to diagnose and report. However, two major health consequences appear to be associated with the ZIKV outbreak which sets it apart from other flaviviruses such as West Nile Virus and Dengue; namely; (a) transmission from an infected mother to fetus resulting in reports of microcephaly in fetuses; and, (b) Guillain-Barre syndrome (GBS) in adults. Several teams have now developed qRT-PCR assays to detect ZIKV. However such tests are relatively expensive, require well-equipped laboratories with specialized equipment, and the procedure takes at least 3 hours to finish. There is an urgent need for a rapid, sensitive, specific and economical diagnostic test for ZIKV. Such an assay could be routinely used in resource-poor settings as well as in doctors' offices, including as part of regular prenatal care. Therefore, the goal of the SBIR Phase I project is to use loop mediated isothermal nucleic acid amplification (LAMP) to develop a rapid, sensitive point of care diagnostic for ZIKV. This technology is of low complexity, requiring only a water bath. Colorimetric results are visible to the naked eye in one hour or less. We will use serial dilutions of ZIKV and other viruses (including West Nile virus, Dengue viruses, and Chikungunya virus) spiked in human blood, saliva, and urine to determine the assay's sensitivity and specificity. Based on our laboratory's extensive previous experience with LAMP assay development, we forecast a lower limit of detection of 10-100 viral genomes, with very high specificity. In addition to rapid, colorimetric ZIKV detection, we will also develop a rapid, lateral flow assay to facilitate the differential analysis with other flaviviruses. The development of rapid point-of-care assays will reduce the dependence on central laboratory testing facilities for epidemiologic surveillance and clinical diagnosis, a key advantage in the resource-poor areas where the epidemic is currently prevalent. Further, we anticipate that the availability of a rapid test will result in the incorporation of ZIKV testing into the sustainable, routine evaluation of women who are pregnant or anticipating pregnancy, as well as their partners.