Wastewater-based epidemiology (WBE), also known as wastewater monitoring or surveillance, has been demonstrated as a promising, early warning, and unbiased tool for monitoring SARS-CoV-2 in wastewater. This environmental approach is based on the assumption that anything emitted by humans and stable in wastewater may be utilized to determine the initial concentration expelled by a target population. That said, testing wastewater for SARS-CoV-2 provides a real depiction of the viral prevalence within a community. In other words, WBE provides SARS-CoV-2 concentration data which can be used to assess infection trends, identify hotspots, as well as inform health authorities and decision makers regarding COVID-19 epidemiological situation. More than that, studies have found that increases in SARS-CoV-2 viral concentration in wastewater can precede increases in clinical infected cases by days to weeks. WBE therefore provides a complementary technique of SARS-CoV-2 surveillance, which is independent of clinical testing and, in many cases, outperforms it. Despite being promising, such surveillance approach faces some challenges associated mainly with quantifying SARS-CoV-2 viral RNA concentration in wastewater. While the quantitative reverse transcription polymerase chain reaction (RT-qPCR) has been the most commonly used assay for quantification, this test comes with its own challenges, one being the presence of inhibitors, and the other being the fact it is time-consuming. Moreover, this assay is the same used for clinical testing, which brings up a major issue due to limited access to RT-qPCR consumables that most laboratories use. Droplet digital reverse transcription polymerase chain reaction (RT-ddPCR) is another assay used for SARS-CoV-2 quantification. This assay offers higher sensitivity and specificity, a better tolerance to inhibitors, as well as an absolute quantification without the need of a standard curve compared to qPCR. However, all these advantages are offset by its high operating cost. Thus, it is imperative to find alternative assays that are more robust than qPCR and more affordable than ddPCR. One such possibility is the loop-mediated isothermal amplification (LAMP) assay, a well-demonstrated nucleic acid procedure that was first developed in 2000. It was reported to be cheaper, specific and highly sensitive. The amplification mechanism of the LAMP assay relies on auto-cycling strand extension with loop formation where nucleic acid is amplified under isothermal conditions, while eliminating the need for thermal cyclers. Another thing that makes LAMP assay interesting is the use of four to six independent primers annealing which makes it reach high specificity and allows effective multiplex applications. However, the use of RT-LAMP for WBE is very limited. Therefore, if implemented, this approach will significantly reduce the time and costs associated with WBE and be a highly useful tool in the surveillance of SARS-CoV-2 and even other infections in the future.
To test the presence of SARS-CoV-2 by RT-LAMP, we used the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit (Cat No. E2019S) from New England BioLabs (Ipswich, MA, USA), which is a 30-min 65°C colorimetric assay. The kit comes with WarmStart Colorimetric 2X Master Mix with UDG, SARS-CoV-2 Positive Control (targeting the N gene), Internal Control primer Mix (targeting human RNA rActin), SARS-CoV-2 LAMP Primer Mix (targeting N and E genes), Guanidine Hydrochloride, and Nuclease-free Water. Following the instructions manual, we run six different tubes: no template control (NTC), positive control, internal control with positive SARS-CoV-2 RNA, internal control with negative SARS-CoV-2 RNA, sample test with positive SARS-CoV-2 sample RNA, and last a sample test with negative SARS-COV-2 sample RNA. After 30 min at 65°C using a qPCR machine, a colorimetric change to yellow or orange is used to report the results, which enables the outcome of viral RNA amplification to be read by naked eye without the need of sophisticated instruments.
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