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Wastewater samples are often very diluted large volume samples and need to be concentrated in order to extract the virus. Several methods are currently used including ultracentrifugation, PEG precipitation, membrane filtration and magnetic beads – and we have a workflow that works for all of them.
Download our app note to find out more about each method
One of the fastest, most efficient ways to concentrate viral RNA in wastewater is to use charged magnetic beads. After an initial gentle centrifugation to remove debris from the wastewater, Dynabeads are added to the supernatant to bind the RNA. The beads are then separated out using a magnet and the RNA eluted from them. Beads offer streamline sample concentration that is easy to automate and almost 100% efficient.
- Suitable for up to 5-50ml of wastewater sample volumes
- 1.5 hours from sample to RNA
Ultracentrifugation of wastewater samples, at 150,000 x g for 45 min at 4°C, pellets viral particles and nucleic acids concentrating the viral RNA and removing low-density contaminants from wastewater samples, in a rapid and scalable way.
Our method uses a sucrose cushion and the Thermo Scientific Sorvall WX+ Ultracentrifuge with the Thermo Scientific SureSpin 632 Swinging-Bucket Rotor. (The SureSpin 632 Swinging-Bucket Rotor is recommended as a substitute rotor with increased performance).
- Suitable for 20ml wastewater sample volumes (scalable)
- 4.5 hours from sample to RNA
- Enables detection and/or quantification of SARS-CoV-2 in low-prevalence areas (as low as 1 positive test per 10,000 inhabitants)
After an initial gentle centrifugation to remove debris, the wastewater supernatant is filtered through a 0.22 μm filter and precipitated with PEG (8% w/v, 16 g) and NaCl (0.5 M, 5.844 g) overnight at 4 °C. Precipitated filtrates were then centrifuged at 16,900 g for 30 minutes at 4 °C, supernatants poured off, and pellets resuspended in 2 mL 1X PBS solution. The Thermo Scientific Sorvall LYNX 6000 Superspeed Centrifuge and Thermo Scientific Fiberlite F12-6 x 500 LEX rotor are ideal for this method.
- Suitable for up to >200ml of wastewater sample volumes
- 24 hours from sample to RNA
After an initial gentle centrifugation to remove debris, the wastewater supernatant is filtered through a 0.45μm pore size filter to bind RNA/virus. The filter membrane is removed from the device, RNA can be eluted off the filter and collected prior to extraction. Thermo Scientific centrifuges and Nalgene Analytical filters provide proven quality and security for filtering wastewater samples.
- Suitable for up to 200ml of wastewater sample volumes
- 1-1.5 hours from sample to RNA
One of the fastest, most efficient ways to concentrate viral RNA in wastewater is to use charged magnetic beads. After an initial gentle centrifugation to remove debris from the wastewater, Dynabeads are added to the supernatant to bind the RNA. The beads are then separated out using a magnet and the RNA eluted from them. Beads offer streamline sample concentration that is easy to automate and almost 100% efficient.
- Suitable for up to 5-50ml of wastewater sample volumes
- 1.5 hours from sample to RNA
Ultracentrifugation of wastewater samples, at 150,000 x g for 45 min at 4°C, pellets viral particles and nucleic acids concentrating the viral RNA and removing low-density contaminants from wastewater samples, in a rapid and scalable way.
Our method uses a sucrose cushion and the Thermo Scientific Sorvall WX+ Ultracentrifuge with the Thermo Scientific SureSpin 632 Swinging-Bucket Rotor. (The SureSpin 632 Swinging-Bucket Rotor is recommended as a substitute rotor with increased performance).
- Suitable for 20ml wastewater sample volumes (scalable)
- 4.5 hours from sample to RNA
- Enables detection and/or quantification of SARS-CoV-2 in low-prevalence areas (as low as 1 positive test per 10,000 inhabitants)
After an initial gentle centrifugation to remove debris, the wastewater supernatant is filtered through a 0.22 μm filter and precipitated with PEG (8% w/v, 16 g) and NaCl (0.5 M, 5.844 g) overnight at 4 °C. Precipitated filtrates were then centrifuged at 16,900 g for 30 minutes at 4 °C, supernatants poured off, and pellets resuspended in 2 mL 1X PBS solution. The Thermo Scientific Sorvall LYNX 6000 Superspeed Centrifuge and Thermo Scientific Fiberlite F12-6 x 500 LEX rotor are ideal for this method.
- Suitable for up to >200ml of wastewater sample volumes
- 24 hours from sample to RNA
After an initial gentle centrifugation to remove debris, the wastewater supernatant is filtered through a 0.45μm pore size filter to bind RNA/virus. The filter membrane is removed from the device, RNA can be eluted off the filter and collected prior to extraction. Thermo Scientific centrifuges and Nalgene Analytical filters provide proven quality and security for filtering wastewater samples.
- Suitable for up to 200ml of wastewater sample volumes
- 1-1.5 hours from sample to RNA
References
1 Stadler et al (2020), Wastewater Analysis of SARS-CoV-2 as a Predictive Metric of Positivity Rate for a Major Metropolis, medRxiv
2 Ahmed et al (2020), Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers, Journal of Travel Medicine, 1-11
3 Nemudryi et al (2020), Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater, Cell Reports Medicine
For Research Use Only. Not for use in diagnostic procedures.
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