评估比色 RT-LAMP 用于未经处理的废水中 SARS-CoV-2 的筛查。
Evaluation of colorimetric RT-LAMP for screening of SARS-CoV-2 in untreated wastewater.
机构信息
Department of Civil and Environmental Engineering, University of Science and Technology, Republic of Korea; Department of Environmental Research, Korea Institute of Civil Engineering and Building Technology (KICT), Republic of Korea; CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
出版信息
Sci Total Environ. 2024 Jan 10;907:167964. doi: 10.1016/j.scitotenv.2023.167964. Epub 2023 Oct 20.
This study compared reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and three reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays targeting the N and E genes of the SARS-CoV-2 genome for detecting RNA in untreated wastewater samples. RT-qPCR assays exhibited consistent amplification down to 2 × 10 GC/reaction, with greater analytical sensitivity at 2 × 10 GC/reaction by US CDC N1 and US CDC N2 assays. In contrast, RT-LAMP exhibited lower sensitivity, detecting SARS-CoV-2 only at or above 2 × 10 GC/reaction. For SARS-CoV-2 seeded wastewater samples, the US CDC N1 assay exhibited greater analytical sensitivity than the US CDC N2, E_Sarbeco, and RT-LAMP assays. Out of 30 wastewater samples, RT-qPCR detected endogenous SARS-CoV-2 RNA in 29 samples, while RT-LAMP identified 27 positive samples, with 20 displaying consistent amplifications in all three RT-LAMP technical replicates. Agreement analysis revealed a strong concordance between RT-LAMP and the US CDC N1 and E_Sarbeco RT-qPCR assays (κ = 0.474) but lower agreement with the US CDC N2 RT-qPCR assay (κ = 0.359). Quantification of SARS-CoV-2 RNA in positive samples revealed a strong correlation between the US CDC N1 and E_Sarbeco assays, while the US CDC N1 and US CDC N2 assays exhibited weak correlation. Logistic regression analysis indicated that RT-LAMP results correlated with RNA quantified by the US CDC N1 and E_Sarbeco assays, with 95 % limits of detection of 3.99 and 3.47 log GC/15 mL, respectively. In conclusion, despite lower sensitivity compared to RT-qPCR assays, RT-LAMP may offer advantages for wastewater surveillance, such as rapid results (estimated as twice as fast), and simplicity, making it a valuable tool in the shifting landscape of COVID-19 wastewater surveillance. Furthermore, LAMP positive wastewater samples might be prioritized for SARS-CoV-2 sequencing due to reduced analytical sensitivity. These findings support the use of RT-LAMP as a specific and efficient method for screening wastewater samples for SARS-CoV-2, particularly in resource-limited settings.
本研究比较了逆转录环介导等温扩增（RT-LAMP）和针对 SARS-CoV-2 基因组 N 和 E 基因的三种逆转录定量聚合酶链反应（RT-qPCR）检测方法，以检测未经处理的废水样本中的 RNA。RT-qPCR 检测方法在 2×10 GC/反应时表现出一致的扩增，美国 CDC N1 和美国 CDC N2 检测方法在 2×10 GC/反应时具有更高的分析灵敏度。相比之下，RT-LAMP 的灵敏度较低，仅在 2×10 GC/反应或更高时才能检测到 SARS-CoV-2。对于接种 SARS-CoV-2 的废水样本，美国 CDC N1 检测方法比美国 CDC N2、E_Sarbeco 和 RT-LAMP 检测方法具有更高的分析灵敏度。在 30 个废水样本中，RT-qPCR 在 29 个样本中检测到内源性 SARS-CoV-2 RNA，而 RT-LAMP 鉴定出 27 个阳性样本，其中 20 个在所有三个 RT-LAMP 技术重复中均显示出一致的扩增。一致性分析显示，RT-LAMP 与美国 CDC N1 和 E_Sarbeco RT-qPCR 检测方法具有很强的一致性（κ=0.474），但与美国 CDC N2 RT-qPCR 检测方法的一致性较低（κ=0.359）。对阳性样本中 SARS-CoV-2 RNA 的定量分析表明，美国 CDC N1 和 E_Sarbeco 检测方法之间存在很强的相关性，而美国 CDC N1 和美国 CDC N2 检测方法之间相关性较弱。逻辑回归分析表明，RT-LAMP 结果与美国 CDC N1 和 E_Sarbeco 检测方法定量的 RNA 相关，95%检测限分别为 3.99 和 3.47 log GC/15 mL。总之，尽管与 RT-qPCR 检测方法相比灵敏度较低，但 RT-LAMP 可能在废水监测方面具有优势，例如快速结果（估计快两倍）和简单性，使其成为 COVID-19 废水监测不断变化的格局中的一种有价值的工具。此外，由于分析灵敏度降低，LAMP 阳性废水样本可能会被优先用于 SARS-CoV-2 测序。这些发现支持将 RT-LAMP 用作筛查废水样本中 SARS-CoV-2 的特异性和高效方法，特别是在资源有限的环境中。
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