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

Jesmin Akter; Wendy J M Smith; Metasebia Gebrewold; Ilho Kim; Stuart L Simpson; Aaron Bivins; Warish Ahmed

doi:10.1016/j.scitotenv.2023.167964

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

Sci Total Environ. 2024 Jan 10:907:167964. doi: 10.1016/j.scitotenv.2023.167964. Epub 2023 Oct 20.

Authors

Jesmin Akter¹, Wendy J M Smith², Metasebia Gebrewold², Ilho Kim³, Stuart L Simpson⁴, Aaron Bivins⁵, Warish Ahmed⁶

Affiliations

¹ 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.
² CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia.
³ 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.
⁴ CSIRO Environment, Lucas Heights, NSW 2234, Australia.
⁵ Department of Civil & Environmental Engineering, Louisiana State University, Baton Rouge, LA 70803, United States of America.
⁶ CSIRO Environment, Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia. Electronic address: Warish.Ahmed@csiro.au.

PMID: 37865239
DOI: 10.1016/j.scitotenv.2023.167964

Abstract

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.

Keywords: COVID-19; RT-LAMP; RT-qPCR; SARS-CoV-2; Surveillance; Wastewater.

MeSH terms

COVID-19*
Colorimetry
Humans
Molecular Diagnostic Techniques
Nucleic Acid Amplification Techniques
RNA, Viral
SARS-CoV-2* / genetics
Sensitivity and Specificity
Wastewater
Wastewater-Based Epidemiological Monitoring

Substances

Wastewater
RNA, Viral

Supplementary concepts

LAMP assay