RT-qPCR for Detection of SARS-CoV-2 in Wastewater V1

As part of the global response to the 2019 novel Coronavirus (SARS-CoV-2) pandemic, it was determined that SARS-CoV-2 RNA was detectable in the faeces of both symptomatic and asymptomatic patients (1). Further analysis demonstrated that a wastewater epidemiological (WWE) approach, similar to that used to track other viruses (i.e. Poliovirus), could be employed to monitor the spread of SARS-CoV-2. The presence of, or changes in concentration of viral RNA within the wastewater network can assist in monitoring the emergence of further viral peaks (2). Thus, monitoring the spread of Covid-19 using the WWE approach has been extensively explored in several countries (3). Viral RNA is extracted from a known volume of sewage treatment works influent and amplified by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) RT-qPCR is a process whereby RNA is first converted to complementary DNA (cDNA) after which it can undergo amplification and quantification via PCR. The amplification of a non-target sequence, in this case the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), can also be carried out to act as an internal control. This procedure, developed by Scottish Environment Protection Agency (SEPA) based on work in the Corbishley lab, Roslin Institute of the University of Edinburgh, outlines the method for amplification and quantification of the RNA extracted from crude sewage samples, but could equally apply to other RNA extracted from other types of environmental sample. For viral RNA extraction from wastewater, please search for "RNA extraction from wastewater for detection of SARS-CoV-2" by the same authors of this protocol on protocols.io References: 1. Jones, D. L., Baluja, M. Q., Graham, D. W., Corbishley, A., McDonald, J. E., Malham, S. K., Hillary, L. S., Connor, T. R., Gaze , W. H., Moura , I. B., Wilcox, M. H., & Farkas , K. (2020).Shedding of SARS-CoV-2 in feces and urine and its potential role in person-to-person transmission and the environment-based spread of COVID-19.Science of the Total Environment.https://doi.org/10.1016/j.scitotenv.2020.1413644. 2. Fitzgerald, S., Rossi, G., Low, A., McAteer, S., O’Keefe, B., Findlay, D., Cameron, G. J., Pollard, P., Singleton, P. T. R., Ponton, G., Singer, A. C., Farkas, K., Jones, D., Graham, D. W., Quintela-Baluja, M., Tait-Burkard, C., Gally, D., Kao, R., & Corbishley, A.(2021).Site specific relationships between COVID-19 cases and SARS-CoV-2 viral load in wastewater treatment plant influent. Environmental Science and Technology. https://doi.org/10.1021/acs.est.1c05029 3. Wastewater SARS Public Health Environmental Response (W-SPHERE). https://sphere.waterpathogens.org/about