Nuclisens minimag nucleic acid purification system

NoV particles were eluted and concentrated as described in Lewis and Metcalf (1988 (link)). In brief, 5 g NoV-spiked sediment samples were mixed with 15 mL 3% beef extract in 2 M NaNO₃ (pH 5.5) for 30 min and the solid matter was removed by centrifugation at 2500 × g, 10 min. The pH of the eluent was adjusted to 7.5 and incubated in 15% PEG6000 and 2% NaCl overnight at 4°C and centrifuged at 2500 × g for 80 min. The resulting pellet was subject to nucleic acid extraction, using the CTAB-based extraction (detailed above) and two commercial kits, the NucliSENS® MiniMag® Nucleic Acid Purification System (bioMérieux SA, France), and the PowerViral® Environmental RNA/DNA Isolation Kit (MO BIO Laboratories, USA). Nucleic acids were extracted according to manufacturer's protocol and eluted in 50 μL molecular biology grade water. Prior to extraction using the MiniMag® System, samples were incubated in 10 mg/mL proteinase K solution at 37°C for 60 min. No PCI was used when samples were extracted using the PowerViral® Kit. The characteristics of each approach are summarized in Table 1.

Total nucleic acids were extracted from a 0.5-ml aliquot of the concentrates using the NucliSENS miniMag nucleic acid purification system (BioMérieux, France). The final volumes of the nucleic acid solution were 0.05 ml (surface water and sediment) and 0.1 ml (wastewater samples). Norovirus GI (64 (link), 65 (link)) and GII (66 (link), 67 (link)), sapovirus GI (68 (link)), and hepatitis A and E viruses (69 (link), 70 (link)) were targeted in qRT-PCR assays as described elsewhere (71 (link)).