The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)–based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab–to–RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.

由 SARS-CoV-2（严重急性呼吸综合征冠状病毒 2）冠状病毒引起的 2019 年冠状病毒病（COVID-19）大流行是一项重大的公共卫生挑战。检测现有 SARS-CoV-2 感染和评估病毒传播的快速检测至关重要。基于逆转录环介导等温扩增 (RT-LAMP) 的病毒 RNA 检测方法具有简单、可扩展且广泛适用的检测方法的潜力。与基于 RT 定量聚合酶链反应 (RT-qPCR) 的方法相比，RT-LAMP 检测需要在恒温下孵育，从而无需复杂的仪器。在这里，我们测试了一种双色 RT-LAMP 检测方案，使用N基因特异的引物组检测 SARS-CoV-2 病毒 RNA。我们对从接受 COVID-19 检测的个体收集的 768 份咽拭子样本中分离出的剩余 RNA 样本进行了 RT-LAMP 测定。我们确定了 RT-LAMP 测定法检测 SARS-CoV-2 病毒 RNA 的敏感性和特异性。与使用敏感引物组的 RT-qPCR 检测相比，我们发现 RT-LAMP 检测可靠地检测到 SARS-CoV-2 RNA，RT-qPCR 循环阈值 (CT) 数高达 30，灵敏度为 97.5 %，特异性为 99.7%。我们还开发了一种拭子到 RT-LAMP 检测，不需要事先进行 RNA 分离步骤，与 RT-LAMP 检测相比，它保留了出色的特异性 (99.5%)，但显示出较低的灵敏度 (CT < 30 时为 86%)。此外，我们开发了多重测序方案（LAMP 测序）作为诊断验证程序来检测和记录 RT-LAMP 反应的结果。