ICAR-National Institute of High Security Animal Diseases, Bhopal 462022, India.
ICAR-Indian Veterinary Research Institute, Hebbal, Bengaluru 560024, India.
J Virol Methods. 2024 Sep;329:114995. doi: 10.1016/j.jviromet.2024.114995. Epub 2024 Jul 6.
Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.
诊断采用多种模式对于控制和管理由 SARS-CoV-2 引起的 COVID-19 至关重要。然而，在中低收入国家，扩大逆转录-定量聚合酶链反应（RT-qPCR）作为 SARS-CoV-2 检测的金标准仍然具有挑战性。像酶联免疫吸附测定（ELISA）这样具有成本效益和高通量的替代方法可以解决这个问题。我们开发了一种内部 SARS-CoV-2 核衣壳捕获 ELISA，并在 271 个人类（n = 252）、牛（n = 10）和狗（n = 9）鼻咽拭子样本上进行了验证。该 ELISA 的检测限为 195 pg/100 µL 核衣壳蛋白，与相关冠状病毒无交叉反应，确保对 SARS-CoV-2 具有高特异性。使用接收者操作特征曲线分析评估诊断性能，显示诊断敏感性为 67.78%，特异性为 100%。当排除 RT-qPCR Ct 值>25 的阳性临床样本时，敏感性提高到 74.32%。此外，评分者间可靠性分析显示与 VIRALDTECT II 多重 RT-qPCR 试剂盒具有实质性一致性（κ 值= 0.73-0.80），与 CoVeasy™ COVID-19 快速抗原自检具有完美一致性（κ 值= 0.89-0.93）。我们的研究结果表明，内部核衣壳捕获 ELISA 适合人类和动物的 SARS-CoV-2 检测，满足了具有成本效益的大规模筛选所需的敏感性和特异性阈值。
