A pcr-based method for SARS-COV-2 variant detection in wastewater.

The COVID-19 outbreak, caused by the SARS-CoV-2 virus, rapidly evolved into a worldwide pandemic, as declared by World Health Organization on 11th March 2020. The continued spread since early 2020 has resulted in many variants of this virus. Most mutations found along its genome are known as single nucleotide polymorphisms (SNPs) where only one base pair is mutated. Current real-time qualitative polymerase chain reaction (RT-qPCR) protocols, so called gold-standard method, are used to confirm if a person is positive or negative for the virus. There is a lack of available technology for rapid identification of variants. In order to identify the presence of a viral variant it is necessary to perform sequencing. Sequencing is expensive and may take hours to days to complete. Due to its cost, sequencing is widely unavailable in most countries. Even in countries where sequencing is available, like the UK, the number of samples sequenced are less than 10% of total cases due to extremely high cost. Wastewater-based epidemiology (WBE) is a novel approach that would help monitor and possibly revert the current health crisis. It has been reported the presence of SARS-CoV-2 RNA in faeces of infected individuals. This makes it possible to detect and monitor SARS-CoV-2 in wastewater samples, providing a health status report of the population within the catchment. To this context, WBE also enables to monitor the dissemination of variants for early warning of the outbreak within the defined population. In this project, a RT-qPCR method was developed targeting unique SNPs of SARS-CoV-2. This assay uses two probes, both targeting the same sequence, one with the SNP and the other non- SNP. These mutations are found in the N-gene of SARS-CoV-2 viral RNA, a conserved region that contains unique SNPs specific to each variant for differentiation. By using two probes, binding competition occurs, and the differentiation is done by observing an earlier detection with the SNP probe (~6 cycles). In addition to this, this method can be coupled with a melt curve analysis for further confirmation. Currently, there is a lack of available technology for rapid identification of variants of concern within the community. This assay can be implemented for routine WBE. By developing a SNP-PCR assay to detect specific variants of concern using WBE, it would be possible to accurately detect variants. This information provides a comprehensive health report on the population that could possibly help revert the current health crisis.