RNA荧光原位杂交(RNA-fluorescence in situ hybridization, RNA-FISH)技术利用荧光标记的核苷酸探针，通过互补链杂交，对细胞或组织中特定的RNA序列进行检测和定位。由于RNA-FISH产生的阳性信号较弱，需要结合特异性信号放大，提高信噪比。但传统信号放大技术的背景难以消除，无法定量且分辨率低，是RNA-FISH技术应用的巨大障碍。本文基于第3代杂交链反应(hybridization chain reaction version 3.0，HCR v3.0)，利用一对分裂式探针消除非特异杂交背景，并引发荧光信号放大反应，建立了针对肠道病毒A71 (enterovirus-A71, EV-A71) RNA的敏感、特异的FISH检测方法，并将该技术与蛋白免疫荧光(immunofluorescence, IF)检测结合，通过高分辨率激光共聚焦成像，成功地在单个细胞水平上检测了EV-A71感染细胞后病毒RNA与其聚合酶3D蛋白的分布变化和相互作用情况，并对细胞中病毒RNA和3D蛋白进行定量。发现相较于传统定量方法，如逆转录定量聚合酶链反应和免疫印迹，新一代RNA-FISH技术在单个细胞水平上病毒RNA和3D聚合酶的表达情况与群体细胞检测的结果在趋势上有明显差异。这说明，基于杂交链反应的新一代RNA-FISH技术，可以克服群体细胞数量增减掩盖病毒组分变化的缺点，从而真实反映病毒在单个细胞中的变化。

RNA-fluorescence in situ hybridization (RNA-FISH) is widely utilized to detect the specific RNA sequences and its contribution in cells or tissues through the complementary hybridization with the fluorescence-labeled nucleotide probes. Given the weak signal, RNA-FISH would be combined with the specific signal amplification technique to improve the signal-to-background ratio. However, due to the low resolution, the traditional signal amplification technology is difficult to eliminate the high background and can not quantify RNA accurately. It is an obvious obstacle to the application of RNA-FISH. Based on the third generation hybridization chain reaction (HCR v3.0) technology, the half split probes were designed to eliminate the non-specific hybridization background and trigger fluorescence signal amplification. Here, we established the sensitive and specific RNA-FISH based on HCR v3.0 to detect the viral RNA of enterovirus A71 (EV-A71). Furthermore, combining RNA-FISH with immunofluorescence staining technology (IF) and high-resolution confocal laser imaging, we successfully detected and quantified the viral RNA and 3D polymerase of EV-A71 during the viral infection in a single-cell level. We observed that the viral RNA decreased whereas 3D polymerase increased significantly in the late stage of EV-A71 infection. It was apparently different from the traditional quantification by reverse tran-scriptase quantitative polymerase chain reaction(RT-qPCR) and western blot, which were based on the total cells during viral infection. It demonstrates that the new generation of RNA-FISH technology based on hybridization chain reaction can overcome the shortcoming of masking the change of virus composition by the increase or decrease of population cell number, so as to truly reflect the change of virus in a single cell.