Viral nucleic acid purification from water samples

General recommendations for sample preparation

Extraction of viral nucleic acids from water samples often poses several noteworthy challenges. The first problem is that viral titers are generally low, such that large sample volumes must be processed in order to obtain a sufficient level of sensitivity. Furthermore, the varying amounts and sizes of debris suspended in the water column may interfere with sample processing and lead to inconsistent recovery between samples. A third problem is the potential presence of PCR inhibitors such as humic acids. Last but not least, samples must be stabilized to prevent degradation of viral particles in the time between sample collection and purification.

Sample stabilization and viral isolation

Collect ~1 L (+/− 0.5 L) of eluent for samples expected to have a high content of debris and microorganisms, and remove debris by sedimentation. Alternatively, collect ~50 L (+/− 10 L) of clear river or tap water. It is recommended to chill the water samples on ice and to isolate viral nucleic acids within 24 hours.

Adjust the pH of the water to ~3.5 using 2 N HCl. Do not omit this pH-adjustment step as it is required to impart a positive net electric charge on the viral particles. The positive charge is crucial for binding of viral particles to the filter surface in the next step. Pass the pH-adjusted water sample through a 0.45-μm negatively charged filter, such as a mixed cellulose ester membrane (e.g., MF-Millipore, Cat. # HAWP09000). Use of filters with a diameter of 90 mm is recommended to prevent clogging, but the ideal filter diameter may vary depending on the sample volume and the amount of debris within the sample. Viral particles will bind to the surface of the filter and can be directly isolated from the filter without prior elution procedures.

Nucleic acid purification

It is recommended to use the NucleoBond RNA Soil midi kit in combination with the DNA Set for NucleoBond RNA Soil for the isolation of viral nucleic acids from the filter disks.

Cut each filter disk into small pieces and transfer the fragments into a sterile 15-ml centrifuge tube. Add the ceramic beads from four MN Bead Tubes Type A (included with the NucleoBond RNA Soil midi kit) to the 15-ml centrifuge tube along with 25:24:1 Phenol:Chloroform:Isoamyl Alcohol (not included with the kit) and follow the standard kit protocol.

Inclusion of the DNA Set for NucleoBond RNA Soil is required. Because the separation of RNA and DNA is not based on chemical properties but rather on differences in size, large RNA genomes such as the genome of SARS-CoV-2 will elute in the DNA fraction. It is recommended to combine the RNA and DNA eluates before further analysis or to elute RNA and DNA in one combined fraction with DNA Elution Buffer EDNA only. Isolated nucleic acids are free of PCR inhibitors due to a patented inhibitor removal technology.

Alternatively, the NucleoSpin RNA Stool kit can be used to isolate viral nucleic acids from cut filter disks. Put a piece of the cut filter disc into an MN Bead Tube Type A (supplied with the NucleoSpin RNA Stool kit) and follow the instructions in the kit handbook. Follow the protocol for total RNA isolation, using the larger volume of Buffer RST2 for binding and, if isolating DNA in parallel, omit the optional DNA digestion step in the protocol. For high-throughput sample processing, it is recommended to use the NucleoMag DNA/RNA Water kit, following the standard protocol. This kit employs scalable magnetic bead-based purification technology and can be easily automated on many common platforms. Ceramic homogenization beads are available separately in smaller or larger formats for efficient processing of the filter disks (MN Bead Tubes Type A and MN Bead Tubes Type A 5 mL, respectively).

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