RT-LAMP: A Cheaper, Simpler and Faster Alternative for the Detection of SARS-CoV-2 in Wastewater

Abstract

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.

The Detection of SARS-CoV2 Antigen in Wastewater Using an Automated Chemiluminescence Enzyme Immunoassay

“…In addition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) has shown promise and may provide a quick and low-cost technique for observing SARS-CoV-2 in wastewater, as well as amplification within 35 min. Unfortunately, there are limitations to the test's quantification and limit of detection [25]. The GeneXpert system reported by Daidle et al is another fully automated assay used to determine SARS-CoV-2 in wastewater within 37 min.…”

Advances in virus detection methods for wastewater-based epidemiological applications

“…This assay does not require thermal cycling due to the isothermal amplification of the nucleic acid [ 16 ]. A few studies have utilized RT-LAMP assays for rapid detection of SARS-CoV-2 in wastewater samples [ 21 , 22 , 38 , 39 ]. The RT-LAMP-based assays used for the detection of SARS-CoV-2 RNA in sewage samples were colourimetric [ 21 , 38 , 39 ] and fluorescence-based [ 21 ].…”

“…A few studies have utilized RT-LAMP assays for rapid detection of SARS-CoV-2 in wastewater samples [ 21 , 22 , 38 , 39 ]. The RT-LAMP-based assays used for the detection of SARS-CoV-2 RNA in sewage samples were colourimetric [ 21 , 38 , 39 ] and fluorescence-based [ 21 ]. The colourimetric RT-LAMP is based on a colour change due to the decrease in pH caused by the RNA polymerase activity during LAMP reactions.…”

Loop-mediated isothermal amplification-based electrochemical sensor for detecting SARS-CoV-2 in wastewater samples

“…Several groups are currently developing LAMP-based protocols for the detection of SARS-CoV-2 in clinical samples [22] , [23] , [24] , [25] . However, scarce information is reported about RT-LAMP technique as a cheaper and faster option for monitoring the genetic material of SARS-CoV-2 in wastewater-based epidemiology [26] , and its integration with attractive sensing schemes.…”