School of Life Science and Technology, Xidian University, Xi'an, 710071, Shaanxi, People's Republic of China.
Engineering Research Center of Molecular & Neuroimaging, Ministry of Education, Xi'an, 710071, Shaanxi, People's Republic of China.
J Nanobiotechnology. 2022 Jun 11;20(1):269. doi: 10.1186/s12951-022-01470-1.
RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences-a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5' end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3' end.
Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues.
The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening.
RNA 病毒会周期性引发严重人类疾病的大流行，经常造成巨大的经济损失。在这里，我们提出了一种无需核酸提取和扩增的 RNA 病毒检测探针，基于双链分子信标方法，用于敏感和简单地检测猪瘟病毒（CSFV）和严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）。这种 RNA 病毒探针包含两个碱基序列 - 识别链，与 CSFV N2 或 SARS-CoV-2 N 的特定结构域结合，5'端标记有荧光团（FAM），互补链（CSFV-Probe B 或 SARS-CoV-2-Probe B），与 3'端标记的淬灭剂（BHQ2）结合。
使用线性分子信标探针技术，RNA 病毒探针对应 CSFV 和 SARS-CoV-2 的检测限分别低至 0.28 nM 和 0.24 nM。在将 CSFV E2 和 SARS-CoV-2 N 基因转染到相应的宿主细胞后，RNA 病毒探针的监测表明，荧光信号以浓度和时间依赖性方式显著增强。这些结果得到了定量（qRT-PCR）和可视化（共聚焦显微镜）分析的支持。此外，还使用 CSF 阳性猪样本和模拟 SARS-CoV-2 感染的小鼠样本证明了其在不同分布的病毒核酸系列组织中的适用性。
所提出的 RNA 病毒探针可作为一种无 PCR、具有成本效益且快速的即时护理（POC）诊断平台，用于靶标 RNA 病毒检测，为早期大规模病毒筛查方便地监测不同的 RNA 病毒提供了巨大的潜力。
