Evaluation of colorimetric RT-LAMP for screening of SARS-CoV-2 in untreated wastewater

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 × 10² GC/reaction, with greater analytical sensitivity at 2 × 10¹ 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 × 10³ 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 log₁₀ 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.