Jul 19, 2024
Wastewater grab sample processing with PEG-8000 precipitation
- Dilip Abraham1,
- Nirmal kumar1,
- Vinoth kumar1,
- Ganesh Rajamanickam1,
- Venkata Raghava Mohan2
- 1Wellcome Trust Research Laboratory, Christian Medical College Vellore, India;
- 2Department of Community Health and Development, Christian Medical College Vellore, India
Protocol Citation: Dilip Abraham, Nirmal kumar, Vinoth kumar, Ganesh Rajamanickam, Venkata Raghava Mohan 2024. Wastewater grab sample processing with PEG-8000 precipitation . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q3ojl1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2024
Last Modified: July 19, 2024
Protocol Integer ID: 103219
Keywords: Wastewater, PEG-8000, Environmental sample concentration, peg molecule, peg concentration, combination of peg concentration, wastewater sample, rna extraction step, concentration of wastewater sample, viruses in the interpolymer space, isolating virus, peg, aqueous polymer, subsequent qpcr assay, soluble synthetic polymer, extraction, virus, polyethylene glycol, rna extraction, wastewater, qpcr assay, rna
Funders Acknowledgements:
Bill & Melinda Gates Foundation
Grant ID: INV-049093
Abstract
PEG (Polyethylene Glycol) is a chemically inert, nontoxic, water-soluble synthetic polymer and has been used in aqueous polymer two-phase systems that help concentrating and isolating viruses from a variety of environmental samples. PEG is known as a good inductor of attractive interactions that crystallize viruses in the interpolymer spaces between PEG molecules. So, the combination of PEG concentration and RNA extraction steps enable 900– 1500X concentration of wastewater samples and sufficiently eliminate most of the organic matter, which could inhibit the subsequent qPCR assay.
Materials
- PEG 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #2139-1KG
- Sodium Chloride Fisher BioReagents™Fisher ScientificCatalog #BP358-1
- 1X PBS (Phosphate-buffered saline )
- Ethanol Absolute Honeywell Catalog #02875
- Whirl-Pak‱ Sample Bag Merck Catalog #WPB01027WA-500EA
- Nalgene‱ centrifuge bottles, Style 3122 Merck Catalog #B0408-4EA
- SPINIX‱ – Vortex Shaker Tarsons Catalog #3020
- Eppendorf‱ Centrifuge 5910 R Merck Catalog #EP5943000246
- Nalgene Centrifuge Bottle Sealing Cap Assembly Catalog number: DS3131-0038.
Sample
Wastewater grab samples
Procedural steps - PEG 8000 concentration method:
2h 15m
Collect 350 mL of sewage samples in a sterile Whirl-Pak bag and disinfect the sampling bag with ethanol to avoid contamination.
Transfer the bag to the laboratory in a cold chain 4 °C .
The sewage sample is transferred to a sterile 250 mL Nalgene centrifuge bottle.
Add 25 g of PEG 8000 and 5.6 g of NaCl in 250 mL of the sample to get the final concentrations of 10 % (w/v) and 0.3 Molarity (M) respectively.
Vortex the mixture at 500 rpm for 00:15:00 until the reagents are completely dissolved in the suspension.
15m
Thereafter, seal the Nalgene bottles using the cap assembly, and centrifuge the mixture at 12000 x g, 02:00:00 until the pellet is visibly seen.
2h
Resuspend the pellet in 3 mL of PBS (7.4 ). Then, aliquot 1 mL of the suspension into each of 3 screw cap tubes.
The resulting pellet is stored at -20 °C until further process.
Freeze-thawing of primary concentrates is limited to once.
Protocol references
1. Wu F, Zhang J, Xiao A, Gu X, Lee WL, Armas F, et al. SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases. mSystems 2020 Aug 25 [cited 2020 Nov 30];5(4)
2. Kumar M, Patel AK, Shah AV, Raval J, Rajpara N, Joshi M, et al. First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2. Science of The Total Environment. 2020 Dec 1;746:141326