| [34] |
Australia |
(i) electronegative membranes:
-
•
Sample pH adjustment to ∼3.5 to 4
-
•
Sample filtration (100-200 mL) through 0.45-μm-pore-size, 90-mm electronegative membranes
-
•
homogenisation of the samples using a Precellys 24 tissue homogenizer 3 × 20 s at 8000 rpm at a 10 s interval
(ii) ultrafiltration:
-
•
centrifugation (4750×g, 30 min) of wastewater (100-200 mL)
-
•
centrifugation (3500×g, 15 min) using the Centricon® Plus-70 centrifugal filter (10 kDa cut-off)
-
•
the concentrate cup was inverted and placed on top of the sample filter cup
-
•
centrifugation (1000×g, 2 min) and collection of the concentrated sample (∼250 μL)
|
- |
- |
| [35] |
Australia |
(i) adsorption-extraction with three different pre-treatment techniques (A-C):
-
•
acidification of the sample to pH 4.0 (method A), no sample pre-treatment (method B), and addition of MgCl2 (25 mM) (method C)
-
•
filtration of samples through electronegative membranes (0.45-μm pore-size, 47-mm diameter)
-
•
placement of the membrane into a 2 mL-bead beating tube
(ii) Centrifugal filter device methods (D, E):
-
•
Method D: centrifugation (4500×g, 10 min) of the sample at 4 °C; concentration of the supernatant using an Amicon® Ultra-15 centrifugal filter device (molecular weight cut-off 30 kDa); centrifugation (4750×g, 10 min) at 4 °C; collection of the concentrated sample (400 μL); and transportation into a 2 mL-bead beating tube
-
•
Method E: centrifugation (3500×g, 30 min) of the supernatant at 4 °C through the Centricon Plus-70 centrifugal filter device (molecular weight cut-off 10 kDa); collection of the concentrated sample (300 μL) and mixing with 100 μL of DNase and RNase free water and transportation into a 2 mL-bead beating tube; re-suspension of the pellet in 800 μL Trizol; transportation of 400 μL of the concentrated sample to a 2-mL bead beating tube
(iii) PEG precipitation (F):
-
•
centrifugation (10000×g, 20 min) at 4 °C for removal of large particles and debris
-
•
transportation of supernatant to a new centrifuge tube and storage at 4 °C
-
•
recovery of MHV from the pellet; re-suspension of the pellet in beef extract and 0.05 M glycine (pH 9.0) at a 1:5 ratio
-
•
agitation of the pellet on shaking incubator (200 rpm, 30 min)
-
•
centrifugation of the pellet (10000×g, 10 min) at 4 °C andtransfer of the supernatant into the centrifugal tube containing the initial centrifugation step supernatant
-
•
neutralisation of the supernatant mixture using 2 M HCl (2M
-
•
addition of PEG and NaCl to the supernatant at ratios of 10% and 2% w/v, respectively
-
•
incubation of the centrifuge tubes at 4 °C for 2 h on an orbital shaker set to 120 rpm
-
•
centrifugation of the sample (10000×g, 30 min) at 4 °C
(iv) Ultracentrifugation (G):
-
•
sample centrifugation (10000×g, 1 h) at 4 °C
-
•
re-suspension of the pellet in 3.5 mL of 0.25 N glycine buffer (pH 9.5)
-
•
incubation of the sample on ice for 30 min; neutralization of the sample by the addition of 3 mL of 2 × PBS (pH 7.2)
-
•
centrifugation of the supernatant (12000×g, 15 min) at 4 °C
-
•
ultracentrifugation (100000×g 1 h) at 4 °C
-
•
re-suspension of the pellet in 400 μL of 1 × PBS (pH 7.2) and transportation to a 2-mL bead beating tube
|
MHV |
26.7-65.7%, |
| [4] |
Australia |
|
- |
- |
| [7]* |
Brazil |
|
Murine Norovirus |
1.6 – 2.6 % |
| [25]* |
France |
|
- |
- |
| [6]* |
France |
-
•
homogenisation of samples
-
•
centrifugation (200000×g, 1 hour) of 11 mL at 4 °C
-
•
resuspension of viral pellets in 400 μL of PBS 1X buffer
|
- |
- |
| [32]* |
India |
-
•
centrifugation (4500×g, 30 min) of sewage samples (50 mL)
-
•
filtration of supernatant using 0.22 μm filters
-
•
concentration of each filtrate using two the 96 well filter plate and PEG method (mixing of 80 g/L PEG and 17.5 g/L of NaCl in 25 mL filtrate and incubation at 17 °C, 100 rpm centrifugation (13000×g, 90 min) and re-suspension of the supernatant in 300 μL RNase free water)
|
- |
- |
| [27]* |
Israel |
-
•
centrifugation of 0.25-1 L of sewage/wastewater effluent to remove large particles
-
•
concentration of the supernatant with PEG or alum (20 mg/L) precipitation, followed by additional centrifugation
-
•
incubation of the mixture at 4 °C with 100-rpm agitation (12 h) and centrifugation (14000×g, 45 min) at 4 °C to pellet the virus particles
-
•
resuspension of the virus particles in PBS solution
-
•
filtration of the aqueous phase (containing virus particles) through a 0.22 μm filter further concentration with Ultra-15 centrifugal tubes (30 kDa cut-off) to a final volume of 1 mL
|
- |
- |
| [22] |
Italy |
-
•
two-phase (PEG-dextran method) separation (modified WHO poliovirus protocol)
-
•
centrifugation of 250 mL of wastewater to pellet solids
-
•
mixing of clarified wastewater with dextran and PEG (the mixture was left to stand overnight at 4 °C in a separation funnel)
-
•
collection of the bottom layer and the interphase and addition to the pellet from the initial centrifugation
|
- |
- |
| [24]* |
Italy |
-
•
two-phase (PEG-dextran method) separation (modified WHO poliovirus protocol)
-
•
centrifugation of 250 mL of wastewater to pellet solids
-
•
mixing of clarified wastewater with dextran and PEG (the mixture was left to stand overnight at 4 °C in a separation funnel)
-
•
collection of the bottom layer and the interphase and addition to the pellet from the initial centrifugation
|
Alphacoronavirus HCoV 229E |
2.04 ± 0.70% |
| [9]* |
Italy |
|
- |
- |
| [33]* |
Japan |
(i) electronegative membrane vortex method:
-
•
addition of 2 mL and 50 mL of 2.5 M MgCl2 to 200 mL of influent wastewater and 5,000 mL of secondary-treated wastewater samples, resp.
-
•
addition of 50 mL of 2.5 M MgCl2 to 5 L of river water samples
-
•
filtration of wastewater and river water samples through a mixed cellulose ester membrane (pore size 0.8 μm; diameter, 90 mm)
-
•
addition of 10 mL of an elution buffer in a 50-mL plastic tube containing the membrane
-
•
vigorous vortexing of the membrane and addition of 5 mL of elution buffer to obtain a final volume of approx. 15 mL
-
•
centrifugation (2000×g, 10 min) at 4 °C
-
•
filtration (0.45 μm pore size, diameter 25 mm) of the supernatant and concentration using a Centriprep YM-50 ultrafiltration device
(ii) adsorption-direct RNA extraction:
|
- |
- |
| [26] |
Spain |
-
•
inoculation of bio-banked influent (200 mL) and effluent wastewater samples with PEDV and MgV
-
•
pH adjustment to 6.0 and addition of 0.9 N of AlCl3 (1:100)
-
•
readjustment of pH to 6.0 and mixing with an orbital shaker (150 rpm, 15 min)
-
•
centrifugation (1700×g, 20 min)
-
•
re-suspension of pellet in 10 mL of 3% beef extract (pH 7.4)
-
•
mixing of the samples (150 rpm, 10 min)
-
•
centrifugation (1900×g, 30 min)
-
•
pellet resuspension in 1 mL of PBS
|
PEDV strain CV777
MgV |
influent:
PEDV = 11 ± 2.1%
MgV = 11 ± 3.5%
effluent:
PEDV = 3.3 ± 1.6%
MgV = 6.2 ± 1.0% |
[21]*
[38] |
The Netherlands |
-
•
centrifugation (4654×g, 30 min) to remove large particles
-
•
filtration of 100-200 mL supernatant with Centricon® Plus-70 centrifugal ultrafilter (10 kDa cut-off) by centrifugation (1500×g, 15 min)
|
F-specific RNA |
73 ± 50% |
| [28]* |
Turkey |
-
•
centrifugation (3200×g, 45 min) of 250 mL of wastewater influent for removal of large particles
-
•
filtration of 250 mL of supernatant with Amicon® Ultra-15 (10 kDa cut-off) by centrifugation (3200×g, 25-40 min)
-
•
collection of 200-600 μL of concentrate
|
- |
- |
| [29]* |
USA |
-
•
mixing of samples to re-suspend particulates
-
•
20 mL was aliquoted to a 38.5 mL ultracentrifuge tube
-
•
addition of a 12 mL sucrose cushion (50% sucrose in TNE buffer [20 mM Tris-HCl (pH 7.0), 100 mM NaCl, 2 mM EDTA])
-
•
purification of samples by centrifugation (150000×g, 90 and 45 min) at 4 °C using a Sorvall WX Ultra series with a Sorvall Surespin™ 630 rotor
-
•
decantation of the supernatant and resuspension of pellets in 200 μL 1X PBS
-
•
storage of pellets at −20 °C for <12 hours prior to RNA extraction
|
- |
- |
| [30] |
USA |
Method A:
-
•
Ultrafiltration with 250 mL of sample at 3000 g x30 min
-
•
Sample concentration (70 mL) with Centricon Plus-70 via centrifugation (1500 g x15 min)
-
•
Further centrifugation at 1000 g x2 min for collection of 350 μL viral concentrate
Method B:
-
•
Adsorption-elution using an electronegative membrane
-
•
Addition of 2.5 M MgCl2
-
•
Filtration through an electronegative filter
-
•
Removal of Mg ions by passage of 200 mL of 0.5 mM H2SO4
-
•
Elution of viruses with 10 mL of 1 mM NaOH (pH 10.8)
-
•
Eluate was recovered in a tube containing 50 μL of 100 mM H2SO4 and 100 μL of 100 x Tris-EDTA buffer
-
•
Centrifugation of 10 mL sample using Centriprep YM-30 to a final volume of 650 μL
|
- |
- |
| [31]* |
USA |
-
•
filtration through a 0.2 μm membrane to remove bacterial debris
-
•
addition to 40 mL filtrate of 4 g of PEG 8000 and 0.9 g NaCl
-
•
centrifugation (12000×g, 2 hours)
-
•
re-suspension of the viral pellet
|
- |
- |
| [8]* |
USA |
-
•
filtration of wastewater sample (500 mL) through 20 μM, 5 μM and 0.45 μM membrane filters
-
•
concentration to 150-200 μL using Corning Spin-X UF concentrators (100 kDa cut-off)
|
- |
- |
