Table 4.
Detection and quantification of SARS-CoV-2 in wastewater and sludge.
| Reference; Location of WWTPs |
Sample type; Sampling mode; Volume; Storage temperature |
Sample pre-treatment | Concentration method | PCR inhibitor treatment | RNA extraction | Concentration in genome copies/L |
Viability test results, number samples with infectious virus/total number of samples | Detection/quantization/viability analysis methods | |
|---|---|---|---|---|---|---|---|---|---|
| Influent | Effluent | ||||||||
|
Medema et al., 2020; The Netherlands |
Municipal wastewater; Composite sampling (24 h); 250 mL; 4 °C |
Centrifugation | Centrifugal filtration (Centricon Plus-70, MWCO of 10 kDa) | – | RNeasy PowerMicrobe (Qiagen) and Biomerieux Nulisens Kit (Biomerieux) in combination with semi-automated KingFisher mL purification system (Thermo Scientific) |
a | a | a | RT-PCR: (one step) EvoScript RNA Probes Master (Roche); Indirect evaluation by F-specific RNA phages assay |
| (Wu et al., 2020b, Wu et al., 2020a) Massachusetts, USA |
Municipal wastewater; Composite sampling (24 h); n.r.; 4 °C |
Pasteurization (60 °C, 90 min) and filtration (0.2 μm pore size) |
PEG/NaCl, centrifugation | – | TRIzol | 1.04 × 103 | a | a | RT-PCR Reverse transcriptase, NEB RT-qPCR; TaqMan fast advanced master mix, Thermofisher |
|
Nemudryi et al., 2020; Montana, USA |
Municipal wastewater; Composite sampling (24 h); 500 mL; n.r. |
Filtration (5 μm and 0.45 μm pore size) | Centrifugal filtration (Corning Spin-X UF, MWCO of 100 kDa) | – | RNeasy Mini Kit (Qiagen) | 104 | a | a | RT-qPCR (one-step) TaqPath™ 1-Step RT-qPCR Master Mix, CG (ThermoFisher); RT-PCR SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific); Q5 High-Fidelity DNA Polymerase (New England Biolabs) |
|
Ahmed et al., 2020 Australia |
Municipal wastewater; Composite sampling (24 h) and grab sampling; 100–200 mL; 4 °C |
Filtration (0.45 μm pore size) | a. Electronegative membrane filtration (HAWP09000, Merk; pore size of 0.45 μm) b. Centrifugal filtration (Centricon Plus-70, MWCO of 10 kDa) |
– | RNeasy PowerWater Kit; RNeasy PowerMicrobiome (Qiagen) | 1.90 × 101 to 1.2 × 102 |
a | a | RT-qPCR (one-step) iTaq™ Universal Probes One-Step Reaction Mix (Bio-Rad) |
|
Wurtzer et al., 2020; France |
Municipal wastewater; n.r.; 11 mL; n.r. |
– | Ultracentrifugation | PCR Inhibitor removal resin (Zymo Research) | PowerFecal Pro kit with a QIAsymphony automated extractor (QIAGEN) | 5.00 × 104 to 3.00 × 106 |
a | a | RT-qPCR |
| Wang et al., 2020b, Wang et al., 2020a; China | Hospital sewage; n.r.; n.r.; n.r. |
– | – | – | SARS-CoV-2 nucleic acid detection kit (Shanghai Berger Medical Technology Co., China) | a | a | 0/6 | RT-PCR; Viability in Vero-E6 cells |
| (Randazzo et al., 2020); Spain |
Municipal wastewater; Grab sampling 200 mL; 4 °C |
pH Adjustment at 6; | Precipitation with AlCl3, centrifugation; elution with beef extract (3%, pH 7.4), centrifugation and resuspension in PBS | – | Nucleospin RNA virus Kit (Macherey-Nagel) | 1.48 × 105 to 3.90 × 105 |
Secondary Effluent: 2.51 × 105 Tertiary Effluent: No detection |
a | PrimeScript™ One Step RT-PCR Kit; RT-qPCR diagnostic panel assays (US CDC, 2019-nCoV RUO Kit and the positive control 2019-nCoV_N_Positive Control by Integrated DNA Technologies). |
|
La Rosa et al., 2020; Italy |
Municipal wastewater; Grab sampling; 250 mL; −20 °C |
Pasteurization (57 °C, 30 min) |
PEG-Dextran precipitation | One step PCR Inhibitor removal kit (Zymo Research) | NucliSENS miniMAG semi-automated extraction system (bioMerieux,) | a | a | a | RT-PCR SuperScript III/IV Reverse Transcriptase (ThermoFisher Scientific); Kit Platinum™ SuperFi™ Green PCR Master Mix, Thermo), |
|
Rimoldi et al., 2020; Italy |
Municipal wastewater; Grab sampling; 500 mL; Specific temperature not reported (samples were under refrigeration) |
Filtration (0.7 μm and 0.2 μm nominal pore size) | Not carried out | – | QIAMP VIRAL RNA mini kit (Qiagen, Hilden, Germany) | a | a | Influent: 0/8 Effluent: 0/4 |
Real-Time RT-PCR Viability in Vero E6 cells |
|
Bar Or et al., 2020; Israel |
Municipal wastewater; Composite sampling (24 h); 0.25–1 L; −20 °C or − 80 °C |
Centrifugation | a. PEG or alum precipitation, centrifugation, b. Centrifugal filtration (Amicon, MWCO of 30 kDa) |
– | RNA extraction kit (RNeasy mini kit- QIAGEN and EasyMAG-bioMerieux, France) | Only Cycle Treshold (Ct) numbers were given (i.e. number of cycles required for the fluorescent signal to cross the threshold) |
Only Cycle Treshold (Ct) numbers were given (i.e. number of cycles required for the fluorescent signal to cross the threshold) |
a | RT-qPCR |
| Haramoto et al., 2020 | Municipal wastewater; Grab sampling; Influent: 200 mL Secondary Effluent: 5000 mL; n.r. |
– | Electronegative membrane-vortex method; Adsorption direct RNA extraction method |
– | RNeasy PowerWater Kit (Qiagen) | Not detected | 2.4 × 103 | a | RT-qPCR Nested PCR |
| Zhang et al., 2020 | Municipal wastewater; Grab sampling; 2.0 L; 4 °C |
Centrifugation | PEG 6000/NaCl precipitation | EZ1 virus Mini kit (Qiagen, Germany) |
Not detected (After primary disinfection tank before septic tank) |
0.5 × 103 to 18.7 × 103 (After septic tank with disinfection with sodium hypochlorite) |
a | RT-qPCR | |
| (Alpaslan Kocamemi et al., 2020); Turkey |
Municipal wastewater sludge; Grab sampling; n.r.; n.r. |
Centrifugation; filtration (0.45 and 0.2 μm nominal pore size); pH Adjustment at 7.0–7.2 | PEG 8000/centrifugation | n.r. | Roche MagNA pure LC total nucleic acid isolation kit using Roche MagNA pure LC system (Penzberg, Germany) | Primary Sludge: 1.25 × 104 Waste Activated Sludge: 1.17 × 104 to 4.02 × 104 |
a | RT-qPCR | |
| Peccia et al., 2020 | Municipal wastewater sludge; Grab sampling; 2.5 mL; -80 °C |
Not carried out | Not carried out (Direct addition of sludge to RNA extraction kit) |
n.r. | RNeasey PowerSoil Total RNA kit, Qiagen | Primary Sludge: 1.7 × 106 to 4.6 x 108 |
a | RT-qPCR | |
Note: n.r. Not reported
a
No available quantitative (concentration) or viability data of virus in wastewater or sludge