Institute for Water and Wastewater Technology, Durban University of Technology, P.O. Box 1334, Durban, 4000, South Africa.
Food Environ Virol. 2021 Dec;13(4):447-456. doi: 10.1007/s12560-021-09489-7. Epub 2021 Jul 26.
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.
逆转录环介导等温扩增 (RT-LAMP) 有可能成为一种更便宜、更快的选择，通过基于废水的流行病学来监测 COVID-19 感染。然而，它在 COVID-19 监测中的应用仅限于临床检测。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 方案，并将这些方案应用于四个污水处理厂的废水监测，持续了 4 周。优化后的 RT-LAMP 方案的检测限为 10 拷贝/25 μl 反应，阳性扩增时间为 35 分钟内。在 4 周的监测中，当分析浓度为 92.7(±28.2)ng/μl 的 1 μl 提取 RNA 时，比色法检测到 12.5%的流行率。当 RNA 模板增加五倍时，流行率增加到 44%。荧光 RT-LAMP 的流行率分别为 31%和 47%，起始模板的浓度分别为 92.7(±28.2)ng/μl 和 480(±134.5)ng/μl 的提取 RNA。用液滴数字 PCR 分析时，所有样本均为 SARS-CoV-2 阳性，病毒载量范围为 18.1 至 195.6 gc/ml 的废水。因此，RT-ddPCR 证实了废水中存在病毒 RNA，尽管浓度较低。此外，RT-LAMP 方案可检测低至 20.7 gc/ml 的拷贝数的 SARS-CoV-2 存在于废水样本中。我们的研究结果表明，RT-LAMP 有可能用于检测废水中的 SARS-CoV-2，为基于废水的 COVID-19 和其他病毒感染的流行病学监测提供了一种比 RT-qPCR 或 RT-ddPCR 更便宜、更快的替代方法。
