A comparison of four commercially available RNA extraction kits for wastewater surveillance of SARS-CoV-2 in a college population

Localized wastewater surveillance has allowed for public health officials to gain a broader understanding of SARS-CoV-2 viral prevalence in the community allowing public health officials time to prepare for impending outbreaks. Given variable levels of virus in the population through public health interventions, proper concentration and extraction of viral RNA is a key step in ensuring accurate detections. With many commercial RNA extraction kits and methodologies available, the performance of 4 different kits were evaluated for SARS-CoV-2 RNA detection in wastewater, specifically focusing on their applicability to lower population densities such as those at university campus dorms. Raw wastewater samples were collected at 4 sites on a college campus over a 24 hour period as a composite sample. Included in these sites was an isolation site that housed students that tested positive for Covid-19 via nasopharyngeal swabs. These samples were analyzed using the following kits: Qiagen All Prep PowerViral DNA/RNA kit, New England BioLabs Monarch RNA MiniPrep Kit, and Zymo Quick RNA-Viral Kit, and the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit. All four sites were processed according to the manufacturer’s guidelines. Extractions were then quantified with RT-qPCR one-step reactions using an N2 primer and a linearized plasmid standard. While the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit (also known as the Zymo Environ Water RNA Kit) only recovered approximately 73% (+/- 38%) SARS-CoV-2 RNA compared to the Zymo Quick-RNA Viral kit, it was the most time efficient kit to yield comparable results. This extraction kit had a cumulative processing time of approximately five hours compared, while the other three kits had processing times between approximately 9 and 9.5 hours. Based on the current research, the most effective kits for smaller population densities are pellet based and include a homogenization, inhibitor removal, and RNA preservation step. Highlights - Samples from smaller population densities make concentrating RNA vital for detection - Pelleting may provide for more timely concentration and extraction of SARS-CoV-2 RNA - RNA shields and PCR inhibitor removal may increase detection of RNA during RT-qPCR Abstract Figure