Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.
Point of sampling detection of Zika virus within a multiplexed kit capable of detecting dengue and chikungunya.
复制标题
DOI：
10.1186/s12879-017-2382-0
复制
发表时间：
2017-04-20
影响因子：
3.7
通讯作者：
Benner SA
中科院分区：
文献类型：
--
作者：
Yaren O;Alto BW;Gangodkar PV;Ranade SR;Patil KN;Bradley KM;Yang Z;Phadke N;Benner SA
寨卡病毒、登革热和基孔肯雅热是三种具有重叠传播媒介的蚊媒病毒。它们引起的疾病在人类患者中具有相似的症状，但需要不同的直接管理步骤。因此，需要快速（<1小时）区分患者样本和捕获的蚊子中的这三种病毒。对速度的需求排除了任何需要复杂的前期样品制备（例如从样品中提取核酸）的测定。在稳健的采样点测定中还排除了扩增子混合物的下游释放，因为这有可能污染未来的样品，从而产生假阳性。报告的程序是直接检测尿液和血浆（用于病人诊断）或压碎的蚊子尸体（用于环境监测）。将尸体捕获在携带季铵基团的纸样品（Q-纸）上，其可直接引入测定中。为了避免PCR的时间和仪器要求，该程序使用环介导等温扩增（LAMP）。下游检测在密封管中进行，在LAMP中使用dTTP-dUTP混合物和不耐热的尿嘧啶DNA糖基化酶（UDG）;这提供了第二种防止前向污染的机制。逆转录LAMP（RT-LAMP）试剂以干燥方式分发，不需要连续的冷藏链。该测试检测未经处理的尿液和其他生物样本中的病毒RNA，区分尿液中的寨卡病毒，基孔肯雅病毒和登革热病毒以及感染活寨卡病毒和基孔肯雅病毒的蚊子。检测限（LOD）为寨卡病毒约0.71 pfu当量病毒RNA，登革热病毒约1.22 pfu当量病毒RNA，基孔肯雅病毒约38个拷贝。一个手持的，电池供电的设备与橙子过滤器被构造成可视化的输出。初步数据显示，这种架构与预先准备的装有冻干试剂/酶混合物的试管一起工作，并且在没有冷藏链的情况下运输，也与人类血浆样本一起工作，以检测印度浦那的基孔肯雅病和登革热。现在有一个带有可视化设备的工具包可用于寨卡病毒、基孔肯雅病毒和登革热的采样点检测。测定输出以ca读取。通过可视化（人眼）三色编码的荧光信号30分钟。干燥形式的测定允许其在低资源环境中运行。本文的在线版本（doi：10.1186/s12879-017-2382-0）包含补充材料，可供授权用户使用。
Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed. The need for speed precludes any assay that requires complex up-front sample preparation, such as extraction of nucleic acids from the sample. Also precluded in robust point-of-sampling assays is downstream release of the amplicon mixture, as this risks contamination of future samples that will give false positives. Procedures are reported that directly test urine and plasma (for patient diagnostics) or crushed mosquito carcasses (for environmental surveillance). Carcasses are captured on paper samples carrying quaternary ammonium groups (Q-paper), which may be directly introduced into the assay. To avoid the time and instrumentation requirements of PCR, the procedure uses loop-mediated isothermal amplification (LAMP). Downstream detection is done in sealed tubes, with dTTP-dUTP mixtures in the LAMP with a thermolabile uracil DNA glycosylase (UDG); this offers a second mechanism to prevent forward contamination. Reverse transcription LAMP (RT-LAMP) reagents are distributed dry without requiring a continuous chain of refrigeration. The tests detect viral RNA in unprocessed urine and other biological samples, distinguishing Zika, chikungunya, and dengue in urine and in mosquitoes infected with live Zika and chikungunya viruses. The limits of detection (LODs) are ~0.71 pfu equivalent viral RNAs for Zika, ~1.22 pfu equivalent viral RNAs for dengue, and ~38 copies of chikungunya viral RNA. A handheld, battery-powered device with an orange filter was constructed to visualize the output. Preliminary data showed that this architecture, working with pre-prepared tubes holding lyophilized reagent/enzyme mixtures and shipped without a chain of refrigeration, also worked with human plasma samples to detect chikungunya and dengue in Pune, India. A kit, complete with a visualization device, is now available for point-of-sampling detection of Zika, chikungunya, and dengue. The assay output is read in ca. 30 min by visualizing (human eye) three-color coded fluorescence signals. Assay in dried format allows it to be run in low-resource environments. The online version of this article (doi:10.1186/s12879-017-2382-0) contains supplementary material, which is available to authorized users.