Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope
Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope
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DOI：
10.1038/s41596-020-00460-7
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发表时间：
2021-01-29
期刊：
影响因子：
14.8
通讯作者：
Yoon, Jeong-Yeol
中科院分区：
文献类型：
--
作者：
Chung, Soo;Breshears, Lane E.;Yoon, Jeong-Yeol
诺如病毒是一种广泛的公共卫生威胁，并且传染剂量非常低。该协议展示了使用基于智能手机的定制荧光显微镜和纸质微流控芯片对水样中的诺如病毒进行极其灵敏的移动检测。抗体偶联的荧光颗粒发生免疫凝集，并通过毛细管作用散布在纸质微流控芯片上，以便使用基于智能手机的荧光显微镜进行个体计数。使用基于强度和尺寸的阈值分析智能手机图像，以消除背景噪声和自发荧光以及分离免疫凝集颗粒。所得颗粒像素计数与测试样本的诺如病毒浓度相关。该协议为智能手机和纸质检测的构建和优化提供了详细的指南。此外，还提供了一个 3D 打印外壳，可将所有组件集成在黑暗环境中。提出了芯片上浓度和更高浓度的测定，以进一步拓宽测定范围。该方法是第一个使用低成本材料作为诺如病毒检测的高灵敏度移动平台提出的。准备好所有材料和试剂后，一次标准测定只需 20 分钟。尽管所描述的方法用于检测诺如病毒，但相同的方案可以适用于通过使用不同的抗体来检测其他病原体。
Norovirus is a widespread public health threat and has a very low infectious dose. This protocol presents the extremely sensitive mobile detection of norovirus from water samples using a custom-built smartphone-based fluorescence microscope and a paper microfluidic chip. Antibody-conjugated fluorescent particles are immunoagglutinated and spread over the paper microfluidic chip by capillary action for individual counting using a smartphone-based fluorescence microscope. Smartphone images are analyzed using intensity- and size-based thresholding for the elimination of background noise and autofluorescence as well as for the isolation of immunoagglutinated particles. The resulting pixel counts of particles are correlated with the norovirus concentration of the tested sample. This protocol provides detailed guidelines for the construction and optimization of the smartphone- and paper-based assay. In addition, a 3D-printed enclosure is presented to incorporate all components in a dark environment. On-chip concentration and the assay of higher concentrations are presented to further broaden the assay range. This method is the first to be presented as a highly sensitive mobile platform for norovirus detection using low-cost materials. With all materials and reagents prepared, a single standard assay takes under 20 min. Although the method described is used for detection of norovirus, the same protocol could be adapted for detection of other pathogens by using different antibodies.