TITLE: Concentration of viruses from sewage using HA filters
AUTHORS: Adélaïde Roguet, Shuchen Feng

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Concentration of viruses from sewage using HA filters</span><span style = "font-weight:bold;"></span></div></div>

[STEPS]
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Take the sample out from the refrigerator.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Homogenize the sample thoroughly, avoiding foaming.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Use a pipette controller, transfer 25 mL of the sample into a 50-mL tube (where MgCl2 has been previously added). Repeat for each replicate. Save the rest of the sample at 4ºC for archive or until decontamination and disposal.
[final concentration]
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Spike in each replicate 5 µL of Bovine Coronavirus recovery control (~100,000 cp/uL), prepared as Bovine coronavirus solution prepared according to http://dx.doi.org/10.17504/protocols.io.bpg8mjzw
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Put the 50-mL tubes on ice until further processing.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Filter the samples in the 50-mL tubes:
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Rinse the clean filtration funnel with sterile deionized water.
Make sure some water is still on the porous plate.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Place the HA filter in the filtration funnel. filter
Make sure the filter gets wet due to the residual water on the porous plate.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Homogenize the sample in the 50-mL tube. Avoid as much as possible the liquid to touch the lid.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Filter the content in the 50-mL tube through a 0.80 µm 47mm cellulose ester filter at low vacuum pressure (~50 mmHg VAC).
?. [Filtration of the sewage samples (in the biosafety cabinet)]
As soon as the filtration is complete, transfer the filter to a 2-mL ZR BashingBead Lysis Tubes containing 400 µL of CTAB buffer solution (previously added). Transfer
It is important not to “over-dry” the filter on the filtration system.
Fold the filter as shown in Figure 1. Do not crease the filters with the forceps.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Put the 2-mL tubes on ice until further processing.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Repeat Steps 6 for the other replicates.
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Store filters at -20ºC until all samples are processed
?. [Filtration of the sewage samples (in the biosafety cabinet)]
Store filters at -80ºC for a minimum of 1h or overnight prior to the nucleic acid extraction. Archive filters at -80ºC.
?. [Nucleic acid extraction]
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?. [Quantification]
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