Ultrafiltration
Concentration of viral particles is essential for efficient nucleic acid isolation from water samples. Explore our recommendations about how to combine the ultrafiltration method for wastewater concentration with our NucleoMag DNA/RNA Water, the NucleoSpin RNA Virus or the NucleoSpin RNA Stool kit for nucleic acid extraction.
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Ultrafiltration – Step by Step protocol recommendation for NucleoMag DNA/RNA Water
The following step by step protocol describes the procedure for a 40 mL wastewater sample. The ultrafiltration method can be adapted to larger volumes. However, an additional pre-filtration step using a 3.5 µm filter is recommended. For larger samples volumes (> 1 Liter), tangential flow filtration is advantageous.
Full technical note with application data
| Ultrafiltration procedure | ||
| 1 | Pellet larger particles | Centrifuge wastewater at 4600 - 4700 x g for 30 min at 4 °C to pellet larger particles or debris. Transfer the supernatant to a fresh tube or container. |
| 2 | Filtration | Filter the cleared wastewater sample through 0.2 µm filter unit or syringe filter to remove residual cellular debris. |
| 3 | Concentration | Concentrate the cleared and filtered wastewater sample using a suitable ultracentrifugation unit (e.g. Centricon® Plus-70 centrifugal ultrafilter units 10 kDa, Millipore; Cat. No.: UFC701008) and recover the wastewater concentrate according to the manufacturer's instructions. |
| 4 | Lysis | Transfer 200 µL of the wastewater concentrate, add 200 µL MWA1 and 10 µL Liquid Proteinase K. Incubate at 56 °C for 10 min. |
| 5 | Binding | Add 25 µL NucleoMag B-Beads and 475 µL Binding Buffer MWA2 and proceed with step 3 of the standard protocol. |
Application data
Detection of MS2 bacteriophage RNA in wastewater concentrates
Wastewater concentrates were generated from 40 mL of a substitute wastewaster samples using ultrafiltration. MS2 bacteriophage RNA was spiked into the concentrates in a dilution series (n = 2 for each dilution) and isolated using the NucleoMag DNA/RNA Water kit (see step-by-step protocol). qRT-PCR analysis was performed with a Taqman probe for MS2 RNA using the SensiFast™ Probe One-Step Lo-ROX kit from Bioline on an Applied Biosystems 7500 Real-Time PCR System. MS2 bacteriophage RNA was detected consistently and reliably over a dilution series with excellent linearity (R² = 0.9999).
Ultrafiltration - Solutions for NucleoSpin RNA Stool and NucleoSpin RNA Virus
NucleoSpin RNA Stool
The ultrafiltration approach for viral concentration has been described by Ahmed et al. 2020 and Medema et al. 2020 utilizing ultrafiltration and 100–200 mL wastewater samples. Larger particles such as debris or bacteria are removed by centrifugation (e.g. 4600 - 4700 x g, 30 min). It is also possible to use standard filtration methods to remove debris. The supernatant (centrifugation) or cleared sample (standard filtration) is subjected to ultrafiltration (e.g. Centricon® Plus-70 centrifugal ultrafilter units 10 kDa, Merck; 1500 - 3500 x g for 15 min).
Recent publication for NucleoSpin RNA Stool
NucleoSpin RNA Virus
The ultrafiltration methode described by Ahmed et al. 2020 and Mederna et al. 2020 can be applied together with the NucleoSpin RNA Virus Kit.
Read more about the NucleoSpin RNA Virus kit
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RNA isolation from wastewater - latest references