Centre for Food Safety, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Dublin, Ireland.
School of Veterinary Medicine, University College Dublin, Dublin, Ireland.
Front Public Health. 2022 May 3;10:863862. doi: 10.3389/fpubh.2022.863862. eCollection 2022.
The diagnostic protocol currently used globally to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is RT-qPCR. The spread of these infections and the epidemiological imperative to describe variation across the virus genome have highlighted the importance of sequencing. SARS-CoV-2 rapid antigen diagnostic tests (RADTs) are designed to detect viral nucleocapsid protein with positive results suggestive of the presence of replicating virus and potential infectivity. In this study, we developed a protocol for recovering SARS-CoV-2 RNA from "spent" RADT devices of sufficient quality that can be used directly for whole virus genome sequencing. The experimental protocol included the spiking of RADTs at different concentrations with viable SARS-CoV-2 variant Alpha (lineage B.1.1.7), lysis for direct use or storage. The lysed suspensions were used for RNA extraction and RT-qPCR. In parallel, we also tested the stability of the viral RNA in the RADTs and the RNA extracted from the RADTs was used as a template for tiling-PCR and whole virus genome sequencing. RNA recovered from RADTs spiked with SARS-CoV-2 was detected through RT-qPCR with C values suitable for sequencing and the recovery from RADTs was confirmed after 7 days of storage at both 4 and 20°C. The genomic sequences obtained at each time-point aligned to the strain used for the spiking, demonstrating that sufficient SARS-CoV-2 viral genome can be readily recovered from positive-RADT devices in which the virus has been safely inactivated and genomically conserved. This protocol was applied to obtain whole virus genome sequence from RADTs ran in the field where the omicron variant was detected. The study demonstrated that viral particles of SARS-CoV-2 suitable for whole virus genome sequencing can be recovered from positive spent RADTs, extending their diagnostic utility, as a risk management tool and for epidemiology studies. In large deployment of the RADTs, positive devices could be safely stored and used as a template for sequencing allowing the rapid identification of circulating variants and to trace the source and spread of outbreaks within communities and guaranteeing public health.
目前全球用于识别严重急性呼吸系统综合症冠状病毒 2(SARS-CoV-2)感染的诊断方案是 RT-qPCR。这些感染的传播以及描述病毒基因组变异的流行病学必要性突出了测序的重要性。SARS-CoV-2 快速抗原诊断测试(RADTs)旨在检测病毒核衣壳蛋白,阳性结果提示存在复制病毒和潜在传染性。在这项研究中,我们开发了一种从“废弃”RADT 设备中回收 SARS-CoV-2 RNA 的方案,这些设备具有足够的质量,可直接用于全病毒基因组测序。实验方案包括在不同浓度下用具有传染性的 SARS-CoV-2 变体 Alpha(谱系 B.1.1.7)对 RADTs 进行加标,然后进行裂解以直接使用或储存。裂解悬浮液用于 RNA 提取和 RT-qPCR。同时,我们还测试了 RADTs 中病毒 RNA 的稳定性,并用 RADTs 提取的 RNA 作为平铺 PCR 和全病毒基因组测序的模板。通过 RT-qPCR 检测到从 RADTs 中回收的 SARS-CoV-2 RNA,C 值适合测序,并且在 4°C 和 20°C 下储存 7 天后从 RADTs 中回收得到了确认。在每个时间点获得的基因组序列与用于加标的毒株对齐,证明可以从已安全失活且基因组保守的阳性 RADT 设备中轻易回收足够的 SARS-CoV-2 病毒基因组。该方案应用于从现场检测到奥密克戎变体的 RADTs 中获得全病毒基因组序列。该研究表明,适合全病毒基因组测序的 SARS-CoV-2 病毒颗粒可以从阳性的废弃 RADTs 中回收,从而扩展其诊断效用,作为风险管理工具和流行病学研究。在 RADTs 的大规模部署中,阳性设备可以安全储存并用作测序模板,从而快速识别循环变体,并追踪社区内暴发的源头和传播,保障公共卫生。
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