Hello, I am new in Environmental virology field. I will like to know the best method for concentration of virus from water and wastewater that do not need Ultra filtration. I have tried using the following protocol;
· Inoculate 2.5M MgCl2 to 100ml sample
· Filter through an HA electronegative filter (pore size: 0.45 μm, diameter: 47 mm; Millipore)
· Pass through 200 ml of 0.5 mM H2SO4 (pH 3) into filter
· Then filter 10 ml of 1 mM NaOH pH 10.5-10.8
· Recover elute in tube containing 0.1ml of 100X TE buffer (pH 8) and 0.1ml of 50 mM H2SO4 (pH 1) ( for neutralization)
Centripep protocol
· Add 10 ml of the elute into Centriprep Concentrator 50 (Nihon Millipore) and centrifuge at 1500 g for 10 min at 4°C.
· Remove the filtrate and rinse the concentrate twice with 10 ml of sterilised MilliQ water by centrifugation at 1500 g for 10 min at 4°C to a final volume of 2 ml.
· Aliquot each concentrate to 200uL final volume
· Store at -80°C.
The following are the problems I encountered.
1. Clogging of filters: For 100ml, I used 2-3 pcs of 0.45μm filter for hours.
2. Do I need to use 0.22μm filter during the concentration and what stage? or only 0.45μm is okay without any bacteria in my concentrate and not losing the viruses in my sample.
Please suggest good method/protocol to use that is not expensive and straight forward. Thanks
InnovaPrep is selling a significant number of Concentrating Pipette instruments for Covid-19 wastewater monitoring. We have a long time user at Hampton Roads Sanitation District that that was the first to begin with this application. Please contact www.innovaprep.com if we can help. thank you. my best,
Hi Abimbola,
do you mean eukaryotic viruses or prokaryotic (phages)? For phages, my colleaugue recently published an optimized protocol, involving optimized buffers, filtration and centrifugation steps, ect. She also had problems with filters clogging. Article Uncovering a hidden diversity: Optimized protocols for the e...
I think the best way is filtration, see e.g., https://www.msrgear.com/blog/water-filter-vs-water-purifier/ . For viruses removal, you may follow https://www.espwaterproducts.com/blog/how-to-remove-viruses-from-water/ as good source of infos.
Evaluating several methods is challenging but our experience has shown precipitation by negatively charged polymers are better. Other considerations include process time, residues, and intangible factors.
So Dr. Juan Eduardo. I bought some mixed cellulose ester membrane from Millipore, but I didn't know how to make an electronegative charged membrane.
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