Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has the potential to become a cheaper and faster option for monitoring COVID-19 infections through wastewater-based epidemiology. However, its application in COVID-19 surveillance has been limited to clinical testing only. We present in this paper two optimized RT-LAMP protocols based on colour change and fluorescence detection and application of these protocols for wastewater monitoring from four wastewater treatment plants over 4 weeks. The optimized RT-LAMP protocols have a limit of detection of 10 copies/25 µl reaction with positive amplification within 35 minutes. Over the 4 weeks of monitoring, the colorimetric protocol detected a prevalence of 12.5%, when 1 µl of extracted RNA with 92.7(± 28.2) ng/µl concentration was analysed. When the RNA template was increased by fivefold, the prevalence increased to 44%. The fluorescent RT-LAMP had a prevalence of 31% and 47% for starting templates of 92.7(± 28.2) ng/µl and 480(± 134.5) ng/µl of the extracted RNA, respectively. All samples were positive for SARS-CoV-2 when analysed with droplet digital PCR, with viral loads ranging from 18.1 to 195.6 gc/ml of wastewater. The RT-ddPCR, therefore, confirms the presence of the viral RNA in the wastewater samples, albeit at low concentrations. Additionally, the RT-LAMP protocols positively detected SARS-CoV-2 in wastewater samples with copies as low as 20.7 gc/ml. The results obtained in our study show the potential application of RT-LAMP for the detection of SARS-CoV-2 in wastewater, which could provide a cheaper and faster alternative to RT-qPCR or RT-ddPCR for wastewater-based epidemiological monitoring of COVID-19 and other viral infections.

逆转录环介导的等温扩增 (RT-LAMP) 有可能成为通过基于废水的流行病学监测 COVID-19 感染的更便宜、更快捷的选择。然而，它在 COVID-19 监测中的应用仅限于临床测试。我们在本文中介绍了两种基于颜色变化和荧光检测的优化 RT-LAMP 协议，以及这些协议在 4 周内对四个废水处理厂的废水监测的应用。优化的 RT-LAMP 方案的检测限为 10 拷贝/25 µl 反应，阳性扩增在 35 分钟内。在 4 周的监测中，当分析 1 µl 提取的 RNA 浓度为 92.7(± 28.2) ng/µl 时，比色方案检测到 12.5% 的流行率。当 RNA 模板增加五倍时，患病率增加到 44%。对于 92.7(± 28.2) ng/µl 和 480(± 134.5) ng/µl 提取的 RNA 的起始模板，荧光 RT-LAMP 的流行率分别为 31% 和 47%。当用液滴数字 PCR 分析时，所有样本均呈 SARS-CoV-2 阳性，病毒载量范围为 18.1 至 195.6 gc/ml 废水。因此，RT-ddPCR 证实了废水样品中病毒 RNA 的存在，尽管浓度很低。此外，RT-LAMP 协议在废水样本中阳性检测到 SARS-CoV-2，其拷贝数低至 20.7 gc/ml。我们研究中获得的结果显示了 RT-LAMP 在检测废水中 SARS-CoV-2 方面的潜在应用，