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. 2020 Jun 24;743:140444. doi: 10.1016/j.scitotenv.2020.140444

Table 3.

Available methods for the concentration of SARS-CoV-2 from wastewater and assays for PCR detection.

Reference Concentration steps PCR assays
(F. Wu et al. 2020)
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    Filtration through 0.22 μm membrane to remove bacterial cells and debris (initial tests revealed little to no viral RNA on filters)

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    PEG precipitation, centrifugation at 12,000g for 2 h or until a pellet was visible
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    US CDC rRT-PCR primer/probe sets targeting three loci of the nucleocapsid protein gene N (CDC, Centers for Disease Control and Prevention)

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    Negative controls: samples from the same wastewater treatment facility taken before the first reported COVID-19 case

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    The rRT-PCR data analysis threshold to call a positive sample included all samples with Ct below 40
(Ahmed et al., 2020)
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    Method A: Direct RNA extraction from the electronegative 0.45 μm filter 90 mm diameter (Ahmed et al., 2015)

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    Method B: Centrifugation at 4750g for 30 min. Supernatant centrifuged at 3500g for 15 min through a centrifugal filter with a cut-off of 10 kDa

 RT-qPCR assays were applied in accordance with the recent literature (Corman et al., 2020; Shirato et al., 2020)
(Medema et al., 2020)
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    Centrifugation step at 4654g for 30 min to remove large particles (debris, bacteria)

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    Supernatant filtered with centrifugal ultrafiltration with a cut-off of 10 kDa at 1500 g for 15 min

 Four primer sets were selected: the N1, N2, N3 sets (US CDC) targetings different regions of the nucleocapsid (N) gene and the set against the envelope protein (E) gene (Corman et al., 2020)
(Wurtzer et al., 2020) Ultracentrifugation at 200,000g for 1 h at 4 °C
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    PCR inhibitor removal resins were used.

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    The RT-qPCR primers were designed within the viral E gene

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    Positive results were confirmed by amplification of a region from the viral RNA dependent-RNA polymerase (Corman et al., 2020)
(La Rosa et al., 2020b) Adaptation of the standard WHO protocol (WHO, 2003) for Poliovirus surveillance for enveloped viruses. Briefly:
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    initial centrifugation of wastewater to pellet the solids

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    mixing of clarified wastewater with dextran and polyethylene glycol (PEG) and left overnight at 4 °C in a separation funnel

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    concentrate was added to the pellet

Chloroform treatment was omitted to preserve the integrity of the enveloped viruses		 RNAs were tested for the presence of SARS-CoV-2 using three different PCR assays:
  • a)

    a broad range of PCR for Coronavirus detection targeting the ORF1ab (Ar Gouilh et al., 2018)

  • b)

    a newly designed primer set specific to SARS-CoV-2

  • c)

    a published nested RT-PCR for SARS-CoV-2 targeting the spike region (Nao et al., 2020)
Randazzo et al. (2020).
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    Concentration method with Al(OH)3 adsorption-precipitation (1 part 0.9 N AlCl3 solution to 100 parts of sample. Incubated for 15 min at room temperature using an orbital shaker)

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    Centrifugation at 1700 g for 20 min

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    Resuspension of pellet in beef extract and centrifugation at 1900 g for 30 min

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    Pellet resuspension in PBS

 RT-qPCR diagnostic panel assays validated by the US Centers for Disease Control and Prevention (CDC, 2020). The first version of the kit with three sets of oligonucleotide primers and probes was used to target three different SARS-CoV-2 regions of the nucleocapsid (N) gene
Authors of this study PEG precipitation
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    Bosphore Novel Coronavirus (2019-nCoV) Detection Kit (Anatolia Geneworks) targeting orf1ab and gene E regions

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    positive samples included all samples with Ct below 40, according to (F. Wu et al. 2020)