Spectrophotometer fluorometer

Nucleic acid was extracted directly from the HA membranes or 150 μL of wastewater sample in the bead-beating tubes using the RNeasy PowerWater Kit (Qiagen). Prior to homogenization, 990 μL of buffer PM1 and 10 μL of β-Mercaptoethanol (Cat. No. M6250–10 mL) (Sigma-Aldrich) were added into each 5 mL bead-beating tube. A known concentration (∼1.5 × 10⁴ gene copies (GC)) of murine hepatitis virus (MHV) was added into each bead-beating tube as an inhibition process control. The bead-beating tubes were then homogenized using a Precellys 24 tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) set for 3 × 15 s at 10,000 rpm at a 10 s interval. After homogenization, the tubes were centrifuged at 4000 g for 4 min to pellet the filter debris and beads. Sample lysate supernatant ranging from 600 to 800 μL in volume was then passed into the extraction to yield purified nucleic acids following the manufacturer's specified protocol. One modification was made: the use of DNase I solution was omitted from the protocol to isolate 100 μL of nucleic acid (i.e., both RNA and DNA). This was done to reduce the sample processing time in the laboratory and for the analysis of DNA and RNA viruses simultaneously. Nucleic acid concentration (ng/μL) were measured using a DeNovix Spectrophotometer & Fluorometer (DeNovix, Wilmington, DE, USA).

Following filtration, using aseptic technique, the membrane was immediately removed, rolled, and inserted into a 5 mL bead-beating tube of the RNeasy PowerWater Kit (Cat. No. 14700-50-NF) (Qiagen, Hilden, Germany) to directly extract nucleic acid from the membrane. Briefly, 990 μL of buffer PM1 and 10 μL of β-mercaptoethanol (Cat. No. M6250-10 mL) (Sigma-Aldrich, St. Louis, Missouri, USA) were added into each bead-beating tube, which was then homogenized using a Precellys 24 tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) set for 3 × 15 s at 10,000 rpm at a 10 s interval. After homogenization, the tubes were centrifuged at 4000 g for 5 min to pellet the filter debris and beads. Nucleic acid extraction was carried out with the RNeasy PowerWater Kit (Qiagen) according to the manufacturer's protocol with two modifications: (i) the use of DNase I solution was omitted from the protocol to isolate nucleic acid (i.e., both RNA and DNA) as respiratory panel included both RNA and DNA viruses; (ii) nucleic acid was eluted with 200 μL of DNase and RNase free water. Sample lysate supernatant ranging from 600 to 800 μL in volume was then used to extract nucleic acid following the manufacturer's protocol. Nucleic acid purity was verified by measuring A_260/280 ratio using a DeNovix Spectrophotometer & Fluorometer (DeNovix, Wilmington, DE, USA).

Immediately after virus concentration, nucleic acid was extracted directly from the HA membranes using the RNeasy PowerWater Kit (Cat. No. 14700-50-NF) (Qiagen, Valencia, CA). Prior to homogenization, 990 µL of buffer PM1 and 10 µL of β-Mercaptoethanol (Sigma-Aldrich; M6250-10 mL) were added into each bead-beating tube. The bead-beating tubes were then homogenized using a Precellys 24 tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, FR) set for 3 × 15 s at 10,000 rpm at a 10 s interval. After homogenization, the tubes were centrifuged at 4,000 g for 5 min to pellet the filter debris and beads. Sample lysate supernatant ranging from 600–800 µL in volume was then used to extract nucleic acid following the manufacturer's specified protocol. Two modifications were made: (i) the use of DNase I solution was omitted from the protocol to isolate nucleic acid (i.e., both RNA and DNA); (ii) nucleic acid was eluted with 200 µL of DNase and RNase free water instead of 100 µL. Nucleic acid purity was verified by measuring 260/280 ratio using a DeNovix Spectrophotometer & Fluorometer (Wilmington, DE, USA).