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Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater.

ABSTRACT: 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 ?6, 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 ?6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest ?6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest ?6 recovery of 6.4% to 35.8%. The comparable ?6 recovery was observed for UF + TRIzol (13.8-30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of ?6 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 ?6 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 ?6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

SUBMITTER: Torii S

PROVIDER: S-EPMC7568484 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater.

Torii Shotaro S Furumai Hiroaki H Katayama Hiroyuki H

The Science of the total environment 20201017

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 virus ...[more]

PMID: 33131851

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Project description:Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

Dataset Information

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater.

Publications

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater.

Similar Datasets

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