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. 2020 Jul 27;8(5):104306. doi: 10.1016/j.jece.2020.104306

Table 2.

Information on the concentration method used for the detection of SARS-CoV-2 RNA in wastewater.

Reference The papers marked with an asterisk (*) were not certified by peer review (preprints) Location given in alphabetical order Main sample concentration steps Surrogate control Recovery
[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)

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[35] Australia
  • addition of 200-μL volume of MHV to a 50-mL aliquot of raw wastewater

(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
  • Adsorption-extraction with electronegative membrane

  • Ultrafiltration with Amicon Ultra-15 centrifugal filter units

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[7]* Brazil
  • Glycine and PEG precipitation

  • Centrifugation using the Centriprep YM 50 filtration system

  • Final volume of 2 mL

Murine Norovirus 1.6 – 2.6 %
[25]* France
  • Vivaspin 50 kDa MWCO filter, down to 500 μLvolume

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[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

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[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)

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[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

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[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

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[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
  • pre-filtration of samples on glass fiber filters (0.7 μm nominal pore size, 145 mm diameter) further filtration on filters (0.2 μm nominal pore size, 145 mm diameter)

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[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:
  • filtration of the water samples with 25 mM MgCl2 through a mixed cellulose-ester membrane (pore size 0.8 μm; diameter 90 mm)

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[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

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[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

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[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

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[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

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[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)

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Abbreviations (given in alphabetical order): MgV: Mengovirus; MHV: Murine hepatitis virus; PBS: Phosphate Buffered Saline; PEDV: Porcine Epidemic Diarrhea Virus; PEG: Polyethelene glycol