Wastewater-Based Epidemiology for Early Detection of SARS-COV-2: Method Development

Utilizing wastewater-based epidemiology (WBE) and quantitative transcription-polymerase chain reaction (qPCR) testing of wastewater (WW) as an effective strategy for public health measures to reduce or mitigate the spread of COVID-19. WBE is an approach that can estimate COVID-19 prevalence in the population by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID, RNA in wastewater in both symptomatic and asymptomatic people. How can SARS-COV-2 RNA be quantitatively measured in environmental wastewater samples using gene markers that yield precision (repeatable), sensitive (detectable), and accurate results? 5. Methods: Create a standard curve or calibration curve that allows the estimate of RNA concentration of environmental samples by comparing them to standards with known RNA concentrations. Achieve this aim by employing four gene markers to reliably detect SARS-CoV-2 RNA in wastewater: two regions in the SARS-COV-2 nucleocapsid N1 and N2 loci of the nucleocapsid (N) gene using primers and probes, attenuated bovine coronavirus (BCoV), and Pepper mild mottle virus (PPMoV), six standard calibration curves were processed using the QuantStudio™ 3 Real-Time PCR System. The purpose of these standard calibration curves was to show that our method and instruments give consistent data with low/no variability from sequence to sequence. Residual calculations show the four gene targets, and the average of their slopes minus each individual slope run is equal to zero, demonstrating no trend in the standard calibration curves or the thermocycler instruments from run to run. There is minimal variability; however, there is no pattern trending upward or downward in a meaningful way. The average for the lower limit of quantification (LLOQ) values is 39, with a standard deviation of 2.87. The LLOQ remains tightly centered around the average. This reveals that the methodology and instruments give consistent data with low variability from sequence to sequence, allowing for confidence in detecting SARS-COV-2. The consistency gives confidence that these four markers (N1, N2, BCOV, PMMOV) can be used. Our laboratory will use this method to test for SARS-COV-2 in environmental wastewater samples.