Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1.
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1.
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DOI：
10.3791/57051
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发表时间：
2018-03-13
期刊：
影响因子：
--
通讯作者：
Benner SA
中科院分区：
文献类型：
--
作者：
Yaren O;Alto BW;Bradley KM;Moussatche P;Glushakova L;Benner SA
寨卡病毒、登革热病毒和基孔肯雅病毒都是通过蚊子传播的，它们引起的疾病与患者的症状相似。然而，它们具有不同的下游患者-患者传播潜力，并且需要非常不同的患者治疗。因此，最近的寨卡疫情使得开发工具迅速区分患者和被困蚊子体内的这些病毒，选择正确的患者治疗方法，并实时了解和管理其流行病学变得迫在眉睫。不幸的是，目前的诊断测试，包括那些获得2016年紧急使用授权和快速通道状态的测试，都是通过逆转录聚合酶链反应（RT-PCR）检测病毒RNA，这需要仪器、训练有素的用户和大量的样品制备。因此，它们必须送到“批准的”参考实验室，这需要时间。事实上，在2016年8月，疾病控制中心（CDC）要求被蚊子叮咬并出现zika指示皮疹的孕妇等待不可接受的2到4周才能得知他们是否被感染。我们非常需要可以在现场进行的测试，资源很少，并且由经过培训但不一定有执照的人员进行。本视频演示了一种符合这些规范的检测方法，可以使用尿液或血清（用于患者）或压碎的蚊子尸体（用于环境监测），所有这些都不需要大量的样品制备。用携带季铵盐基团的纸（q纸）捕获蚊子尸体，然后进行氨处理以管理生物危害。然后不需要RNA分离，直接将它们放入含有冷冻干燥试剂的实验管中，不需要冷藏链。一种改良形式的逆转录环介导的等温扩增与目标特异性荧光标记可置换探针产生读数，在30分钟内，作为三色荧光信号。这是可视的手持，电池供电的设备与橙色过滤器。用密封管和在扩增混合物中dUTP存在的情况下使用耐热性尿嘧啶DNA糖基化酶（UDG）来防止正向污染。
Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time. Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to “approved” reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel. This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.
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影响因子：
7.4
作者：
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通讯作者：
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9.4
作者：
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通讯作者：
Hobbs, Glyn