Yang Zixiao, Zhou Xinrong, Sun Xikui, Cao Liu, Xu Tiefeng, Li Kun, Liu Hongchao, Ji Yanxi, Liu Lihong, Ivanov Konstantin I, Yang Zhonghan, Guo Deyin, Li Chun-Mei
Center for Infection and Immunity, School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, 518107, China; Guangzhou National Laboratory, Guangzhou International Bio-Island, Guangzhou, 510320, China.
Guangzhou National Laboratory, Guangzhou International Bio-Island, Guangzhou, 510320, China; State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510182, China.
Virol Sin. 2025 Aug;40(4):613-623. doi: 10.1016/j.virs.2025.06.006. Epub 2025 Jul 1.
Rapid and accurate detection of infectious virus particles, not just viral nucleic acid, is essential to avoid unnecessary quarantine and effectively control the spread of viral diseases such as coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS). Real-time quantitative polymerase chain reaction (RT-qPCR) was the most widely used detection technique during the COVID-19 outbreak. However, it cannot discriminate between intact infectious viruses and surface-distorted, non-infectious virus particles or naked viral RNA. In this study, we present a strategy for the specific detection of infectious coronaviruses by combining viral receptor capture and reverse transcription loop-mediated isothermal amplification (RT-LAMP). We successfully applied this strategy to detect infectious virus particles of the SARS-CoV-2 surrogate virus and the human coronavirus NL63 (HCoV-NL63). Virus particles were first captured on ELISA plates coated with the recombinant human angiotensin-converting enzyme 2 (hACE2) receptor. Viral RNA was then extracted from the particles and detected by RT-LAMP using virus-specific primers. In our experimental setting, the proposed method had a minimum detection limit (LOD) of 90 PFU/mL, sensitivity of 96.2%, and specificity of 100%. Our study provides a proof-of-concept that viral receptor capture combined with RT-LAMP can differentiate infectious coronaviruses from non-infectious virions or naked viral RNA. This paves the way for this virus detection strategy to become a mainstream tool for the management, prevention and control of epidemic coronavirus diseases.
快速准确地检测传染性病毒颗粒，而不仅仅是病毒核酸，对于避免不必要的隔离并有效控制病毒性疾病（如2019冠状病毒病（COVID-19）、严重急性呼吸综合征（SARS）和中东呼吸综合征（MERS））的传播至关重要。在COVID-19疫情期间，实时定量聚合酶链反应（RT-qPCR）是使用最广泛的检测技术。然而，它无法区分完整的传染性病毒与表面变形的非传染性病毒颗粒或裸露的病毒RNA。在本研究中，我们提出了一种通过结合病毒受体捕获和逆转录环介导等温扩增（RT-LAMP）来特异性检测传染性冠状病毒的策略。我们成功地应用该策略检测了严重急性呼吸综合征冠状病毒2替代病毒和人冠状病毒NL63（HCoV-NL63）的传染性病毒颗粒。病毒颗粒首先被捕获在包被有重组人血管紧张素转换酶2（hACE2）受体的酶联免疫吸附测定（ELISA）板上。然后从颗粒中提取病毒RNA，并使用病毒特异性引物通过RT-LAMP进行检测。在我们的实验条件下，所提出的方法的最低检测限（LOD）为90 PFU/mL，灵敏度为96.2%，特异性为100%。我们的研究提供了一个概念验证，即病毒受体捕获与RT-LAMP相结合可以区分传染性冠状病毒与非传染性病毒体或裸露的病毒RNA。这为这种病毒检测策略成为冠状病毒疫情管理、预防和控制的主流工具铺平了道路。
