Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
John Thomas - Grambling State University, Grambling, LA
Co-Author(s): Lescia Valmond, Grambling State University; Thaddisha James, Grambling State University
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused over one million reported deaths across the United States of America and over 6.5 million reported deaths worldwide. Asymptomatic individuals who do not get tested for COVID-19 have contributed to the transmission of the virus and the gradual decrease in diagnostic testing means that the number of reported cases significantly underestimates the actual disease prevalence. Evidence shows that wastewater surveillance can be used to more accurately follow trends of SARS-CoV-2 in a community (REFERENCE).Weekly wastewater samples were collected from two local sewer sites over a 24-hour period. Samples were centrifuged to separate debris and other materials found in sewage. Polyethylene glycol and NaCl were added to the wastewater supernatant to precipitate the virus (REFERENCE) and a pellet containing viral concentrate was obtained by a second centrifugation. Using the Qiagen QIAmp column extraction kit, RNA was extracted from the viral pellet. Reverse transcription was performed using the Invitrogen High-Capacity cDNA Reverse Transcription Kit. Real-time PCR was performed using the IDT PrimeTime Gene Expression master mix and CDC probes against the N1 and N2 genes to quantify viral genome copies in the wastewater. The N1 and N2 data were normalized to the level of Pepper Mild Mottle Virus (PMMoV) quantified in the wastewater. PMMoV is a virus that infects multiple peppers that humans are likely to consume. During university holidays and times when there are fewer students on campus or in the community, we detected lower levels of the PMMoV.Our data reveals trends in SARS-CoV-2 levels that track with spikes in the U.S. and locally reported cases. With this information, campus administrators, health care providers, epidemiologists, and other decision makers are able to respond when the wastewater data indicates an upward trend. Wastewater-based epidemiology has been used in the past to monitor polio and has gained renewed attention as a useful epidemiological tool. Future work involves continued monitoring of wastewater for SARS-CoV-2 and other pathogens. References:covid.cdc.gov (September 2022)“An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater”,Sci Total Environ. 2021 Sep 1; 785: 147270. Published online 2021 Apr 25. doi: 10.1016/j.scitotenv.2021.147270 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086323/“Pepper mild mottle virus: Agricultural menace turned effective tool for microbial water quality monitoring and assessing (waste)water treatment technologies”, PLoS Pathog. 2019 Apr; 15(4): e1007639. Published online 2019 Apr 18. doi: 10.1371/journal.ppat.1007639
Funder Acknowledgement(s): This research was supported by NSF award #1818697 and the Grambling State University Provost Office.
Faculty Advisor: Paul Kim, Ph.D., Department of Biological Sciences, Grambling State University; Audrey Kim, D.V.M., Ph.D., Department of Biological Sciences, Grambling State University, Kim.email@example.com
Role: Sample CollectingWastewater processingRNA extraction Reverse Transcription PCR