Wastewater grab sample processing with PEG-8000 precipitation
Dilip Abraham, Venkata Raghava Mohan, Nirmal kumar, Vinoth kumar, Ganesh Rajamanickam
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.
Steps
Sample
Wastewater grab samples
Procedural steps - PEG 8000 concentration method:
Collect 350mL 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 250mL Nalgene centrifuge bottle.
Add 25g of PEG 8000 and 5.6g of NaCl in 250mL of the sample to get the final concentrations of 10 (w/v) and 0.3Molarity (M) respectively.
Vortex the mixture at 500rpm for 0h 15m 0s until the reagents are completely dissolved in the suspension.
Thereafter, seal the Nalgene bottles using the cap assembly, and centrifuge the mixture at 12000x g until the pellet is visibly seen.
Resuspend the pellet in 3mL of PBS (7.4). Then, aliquot 1mL 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.
