The Key Laboratory of Molecular Biology of Infectious Diseases designated by the Chinese Ministry of Education, Department of Infectious Diseases, Institute for Viral Hepatitis, Chongqing Medical University, Yuzhong, 400016, Chongqing, China.
College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, Yuzhong, China.
J Nanobiotechnology. 2022 Sep 5;20(1):399. doi: 10.1186/s12951-022-01558-8.
Effective therapeutics and vaccines for coronavirus disease 2019 (COVID-19) are currently lacking because of the mutation and immune escape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on the propagation characteristics of SARS-CoV-2, rapid and accurate detection of complete virions from clinical samples and the environment is critical for assessing infection risk and containing further COVID-19 outbreaks. However, currently applicable methods cannot achieve large-scale clinical application due to factors such as the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods. In this study, we developed an immuno molecular detection method combining capture of the viral spike glycoprotein with monoclonal antibodies and nucleic acid amplification via quantitative reverse transcription PCR to rapidly and accurately detect complete virions.
After constructing a novel pseudovirus, screening for specific antibodies, and optimizing the detection parameters, the assay achieved a limit of detection of 9 × 10 transduction units/mL of viral titer with high confidence (~ 95%) and excellent stability against human serum and common virus/pseudovirus. The coefficients of variation were 1.0 ~ 2.0% for intra-assay and inter-assay analyses, respectively. Compared with reverse transcription-PCR, the immunomolecular method more accurately quantified complete virions. SARS-CoV-2/pseudovirus was more stable on plastic and paper compared with aluminum and copper in the detection of SARS-CoV-2 pseudovirus under different conditions. Complete virions were detected up to 96 h after they were applied to these surfaces (except for copper), although the titer of the virions was greatly reduced.
Convenient, inexpensive, and accurate complete virus detection can be applied to many fields, including monitoring the infectivity of convalescent and post-discharge patients and assessing high-risk environments (isolation rooms, operating rooms, patient living environments, and cold chain logistics). This method can also be used to detect intact virions, including Hepatitis B and C viruses, human immunodeficiency virus, influenza, and the partial pulmonary virus, which may further improve the accuracy of diagnoses and facilitate individualized and precise treatments.
由于严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的突变和免疫逃逸，目前缺乏针对 2019 年冠状病毒病（COVID-19）的有效治疗方法和疫苗。基于 SARS-CoV-2 的传播特性，快速准确地从临床样本和环境中检测到完整的病毒粒子对于评估感染风险和遏制进一步的 COVID-19 爆发至关重要。然而，由于病毒载量高、病毒分离步骤繁琐、实验环境要求高以及实验周期长等因素，目前适用的方法无法实现大规模的临床应用。在本研究中，我们开发了一种免疫分子检测方法，该方法结合了单克隆抗体对病毒刺突糖蛋白的捕获以及通过定量逆转录 PCR 进行的核酸扩增，可快速准确地检测完整的病毒粒子。
在构建了一种新型假病毒后，通过筛选特异性抗体并优化检测参数，该检测方法的检测限达到了 9×10 转导单位/mL 的病毒滴度，具有较高的置信度（95%），并且对人血清和常见病毒/假病毒具有良好的稳定性。内分析和间分析的变异系数分别为 1.0%2.0%。与逆转录-PCR 相比，免疫分子方法更准确地定量了完整的病毒粒子。在不同条件下检测 SARS-CoV-2 假病毒时，SARS-CoV-2/假病毒在塑料和纸上比在铝和铜上更稳定。在这些表面上应用后，完整的病毒粒子可以检测到 96 小时（除铜外），尽管病毒的滴度大大降低。
方便、廉价且准确的完整病毒检测可应用于许多领域，包括监测恢复期和出院后患者的传染性以及评估高风险环境（隔离室、手术室、患者生活环境和冷链物流）。该方法还可用于检测完整的病毒粒子，包括乙型和丙型肝炎病毒、人类免疫缺陷病毒、流感病毒和部分肺病毒，这可能进一步提高诊断的准确性，并有助于实现个体化和精准治疗。
