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An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater.

ABSTRACT: Wastewater-based epidemiology is currently being utilized to monitor the dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on a population scale. The detection of SARS-CoV-2 in wastewater is highly influenced by methodologies used for its isolation, concentration and RNA extraction. Although various viral concentration methods are currently employed, including polyethylene glycol (PEG) precipitation, adsorption-extraction, ultracentrifugation and ultrafiltration, to our knowledge, none of these methods have been standardized for use with a variety of wastewater matrices and/or different kits for RNA extraction and quantification. To address this, wastewater with different physical characteristics was seeded with gamma-irradiated SARS-CoV-2 and used to test the efficiency of PEG precipitation and adsorption-extraction to concentrate the virus from three physiochemically different wastewater samples, sourced from three distinct wastewater plants. Efficiency of viral concentration and RNA extraction was assessed by reverse-transcriptase polymerase chain reaction and the recovery yields calculated. As co-purification of inhibitors can be problematic for subsequent detection, two commonly used commercial master mixes were assessed for their sensitivity and efficiency to detect two SARS-CoV-2 target nucleocapsid (N) gene sequences. Recovery rates varied greatly between wastewater matrices and concentration methods, with the highest and most reproducible recovery rates (46.6-56.7%) observed when SARS-CoV-2 was precipitated with PEG and detected by the Luna® Universal master mix. The adsorption-extraction method was less effective (0-21.7%). This study demonstrates that PEG precipitation is the more robust method, which translates well to varying wastewater matrices, producing consistent and reproducible recovery rates. Furthermore, it is compatible with different kits for RNA extraction and quantitation.

SUBMITTER: Sapula SA

PROVIDER: S-EPMC8086323 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate-phenol-chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF + TRIzol (13.8-30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG + TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV + QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6-21%) among the types of raw sewage. Then, the applicability of modified PEG + TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG + TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

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An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater.

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