Small-Volume Concentration of Viruses from Environmental Water Samples

Résumé

Source: McMinn, B. R. & Korajkic, A. A Small Volume Procedure for Viral Concentration from Water. J. Vis. Exp. (2015)

This video demonstrates a method for concentrating viruses from small volumes of contaminated water using electropositive filtration followed by particle-assisted secondary concentration. It outlines the steps for virus capture, elution, pH-mediated adsorption, and recovery for downstream analysis.

Protocole

1. Tap Water Filtration (Primary Concentration)

1. Measure 100 ml of virus spiked tap water using sterile graduated cylinder.
2. Pour 100 ml of virus spiked tap water into filter housing containing either a 47 mm pleated glass/cellulose filter or a nano-alumina/glass fiber disk filter.
3. Allow water to slowly pass through filter. Apply gentle vacuum to remove remaining residuals of tap water from filter surface.
4. If using trypsin treatment, add 5 ml of 0.25% trypsin to filter surface and incubate for 5 min.
5. Wash trypsin from filter surface using 50 ml of sterile deionized water.

2. Virus Elution from Filter

1. Remove filter housing from 2 L Erlenmeyer flask, and place onto 250 ml side-arm flask.
2. Measure 100 ml of beef extract using graduated cylinder. Slowly pour 100 ml of beef extract into filter housing.
3. Allow beef extract to pour through filter, capturing beef extract in a small (250 ml) Erlenmeyer flask. Apply vacuum to pull through residual beef extract from filter.

3. Celite Secondary Concentration

1. Transfer flasks containing the eluted viruses to a stir plate. Add sterile magnetic stir bar and place on stir plate, mixing to create slight vortex.
2. Add 0.1 g of celite powder to 100 ml of beef extract and allow celite to disperse. Place sterile pH probe into beef extract.
3. Add drop wise 1 M HCl to beef extract to achieve pH 4.0. Allow to slowly mix for 10 min.
4. Place 47 mm pre-filter onto filter housing (as previously described) using a 2 L Erlenmeyer flask.
5. Pour 100 ml beef extract/celite mixture over pre-filter. Allow beef extract to pour through. Apply slight vacuum to pull through residual beef extract from filter.
6. Place metal tube clip on end of filter housing spout. Attach sterile 15 ml polypropylene collection tube to metal tube clip. Place filter housing back into Erlenmeyer flask.
7. Add 5 ml of 1x phosphate-buffered saline (PBS) buffer, pH 9.0 over celite collected on pre-filter.
8. Allow PBS to drip through pre-filter. Apply slight vacuum to pull through residual PBS into 15 ml collection tube.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
Adenovirus 40 stock	ATCC	VR-931
Adenovirus 41 stock	ATCC	VR-930
Sodium Thiosulfate	Fluka Chemical Co.	72051
Celites #577	Fluka Chemical Co.	22142
NanoCeram 47 mm	Argonide	N/A
1MDS 47 mm	3M	6408502
AP-20 Prefilter 47 mm	Millipore Corp.	AP2004700
Sodium Polyphosphate	Acros Organics	390930010
PBS	Sigma	P5368
Hydrochloric Acid	Fisher	A481-212
Difco Beef Extract	BD Biosciences	211520
Stainless Steel Filter Housing	Millipore Corp.	XX2004720