The wastewater was concentrated using the flocculation method using 3.0% w/v beef extract solution (Sigma-Aldrich, St. Louis, USA), pH 9.5 in 0.05 M glycine buffer. Each sewage water (SW1, SW2, and SW3) sample (500 mL) was acidified (Michael-Kordatou et al. 2020 (link)) at pH 3.5 ± 0.1 and 10 mL of the beef extract was agglomerated by the addition of 0.1 M HCl at pH 3.0 followed by stirring for 10 h. The stirred suspension was centrifuged at 10,000×g for 30 min at 4°C. The pellet was dissolved in 8 mL of phosphate buffered saline (PSB) and the RNA was extracted using NucliSENS® miniMAG® system (BioMerieux, Marcy l Etoile, France) according to the manufacturer’s instructions.
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Wastewater Viral RNA Extraction Protocol
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Corresponding Organization : King Saud University
Other organizations : Princess Nourah bint Abdulrahman University, King Abdulaziz City for Science and Technology
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Variable analysis
independent variables
- Concentration of the wastewater using the flocculation method
- PH of the beef extract solution (pH 9.5)
- Acidification of the sewage water samples to pH 3.5 ± 0.1
- Addition of 0.1 M HCl to the beef extract solution to reach pH 3.0
- Stirring duration (10 h)
- Extraction of RNA from the pellet using the NucliSENS® miniMAG® system
- Volume of sewage water samples (500 mL)
- Volume of beef extract solution (10 mL)
- Centrifugation speed (10,000×g)
- Centrifugation duration (30 min)
- Centrifugation temperature (4°C)
- Buffer used for dissolving the pellet (phosphate buffered saline, PBS)
- No positive or negative controls were explicitly mentioned in the provided information.
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