Oct 19, 2020

Public workspaceV.3 - Direct wastewater RNA capture and purification via the "Sewage, Salt, Silica and SARS-CoV-2 (4S)" method V.3

DOI
dx.doi.org/10.17504/protocols.io.bngsmbwe
  • Oscar N Whitney1,
  • Basem Al-Shayeb2,
  • Alex Crits-Cristoph3,
  • Mira Chaplin4,
  • Vinson Fan1,
  • Hannah Greenwald4,
  • Adrian Hinkle4,
  • Rose Kantor4,
  • Lauren Kennedy4,
  • Anna Maurer1,
  • Robert Tjian5,
  • Kara L. Nelson6,
  • UC Berkeley Wastewater-based epidemiology consortium6
  • 1University of California, Berkeley, Tjian & Darzacq laboratory;
  • 2University of California, Berkeley, Banfield & Doudna laboratory;
  • 3University of California, Berkeley, Banfield laboratory;
  • 4University of California, Berkeley, Nelson laboratory;
  • 5University of California, Berkeley, HHMI;
  • 6University of California, Berkeley
  • Coronavirus Method Development Community
Oscar N Whitney
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DOI: dx.doi.org/10.17504/protocols.io.bngsmbwe
Protocol Citation: Oscar N Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium 2020. V.3 - Direct wastewater RNA capture and purification via the "Sewage, Salt, Silica and SARS-CoV-2 (4S)" method. protocols.io https://dx.doi.org/10.17504/protocols.io.bngsmbweVersion created by Oscar N Whitney
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
Created: October 14, 2020
Last Modified: October 19, 2020
Protocol Integer ID: 43250
Keywords: SARS-CoV-2, Wastewater-based epidemiology, Direct capture, RNA extraction, COVID-19,
Abstract
This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.



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Materials
MATERIALS
ReagentTris
ReagentEDTA
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentMicrocentrifuge
ReagentEthanol
ReagentZymo III-P columnZymo ResearchCatalog #C1040-5
ReagentEZ-Vac Vacuum ManifoldZymo ResearchCatalog #S7000
ReagentDurapore® Membrane Filter 5.0 µmMillipore SigmaCatalog #SVLP04700
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentSwinnex Filter HolderMillipore SigmaCatalog #SX0004700
ReagentZymoPURE Elution BufferZymo ResearchCatalog #D4200-7-30
STEP MATERIALS
ReagentZymoPURE Elution BufferZymo ResearchCatalog #D4200-7-30
ReagentTE buffer
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentDurapore® Membrane Filter 5.0 µmMillipore SigmaCatalog #SVLP04700
ReagentSwinnex Filter HolderMillipore SigmaCatalog #SX0004700
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentEZ-Vac Vacuum ManifoldZymo ResearchCatalog #S7000
ReagentZymo III-P columnZymo ResearchCatalog #C1040-5
Protocol materials
ReagentZymo III-P columnZymo ResearchCatalog #C1040-5
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700
ReagentZymoPURE Elution BufferZymo ResearchCatalog #D4200-7-30
ReagentTE buffer
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentMicrocentrifuge
ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700
ReagentZymoPURE Elution BufferZymo ResearchCatalog #D4200-7-30
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentEthanol
ReagentEZ-Vac Vacuum ManifoldZymo ResearchCatalog #S7000
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentTris
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentEZ-Vac Vacuum ManifoldZymo ResearchCatalog #S7000
ReagentZymo III-P columnZymo ResearchCatalog #C1040-5
ReagentEDTA
ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700
ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentEZ-Vac Vacuum ManifoldZymo ResearchCatalog #S7000
ReagentZymo III-P columnZymo ResearchCatalog #C1040-5
ReagentTE buffer
ReagentZymoPURE Elution BufferZymo ResearchCatalog #D4200-7-30
Safety warnings
Wastewater is intrinsically hazardous, so we advise handling wastewater samples in a biosafety cabinet in a BSL2+ laboratory space.
Before start
We developed this procedure to provide a highly efficient, economical, and rapid method for extraction of SARS-CoV-2 RNA from wastewater. Using this procedure at the University of California Berkeley, we have captured and quantified SARS-CoV-2 in the raw wastewater influent of six San Francisco Bay Area treatment plants, as well as at dozens of locations within Bay Area sewersheds. We have also used this method to detect Bacteroides, 18S rRNA, and pepper mild mottle virus (PMMoV) RNA in wastewater, which could serve as indicators of wastewater fecal concentration with which to normalize SARS-CoV-2 concentrations.

This procedure relies on vacuum column processing, which can be performed with a vacuum manifold and vacuum pump or central vacuum line. In our laboratory, this procedure yields concentrated and purified wastewater RNA in less than 3 hours.

In our laboratory, this purification method enables the detection of SARS-CoV-2 N and E gene RNA as well as PMMoV RNA via RT-qPCR probe-mediated detection. Depending on sample origin, we are able to recover an average of 35 ng RNA/mL of purified wastewater sample (min = 9.33 ng/mL, max = 95 ng/mL).
Preparing RNA wash buffers
Preparing RNA wash buffers
PrepareAmount1 L each of two wash buffers - Wash buffer #1 (4S-WB1) and #2 (4S-WB2), for later use during cleanup of RNA bound to silica columns.
4S-WB1 composition:
ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer
NaCl5 M1.5 M300 mL
Ethanol100%20%200 mL
TRIS pH 7.21 M10 mM10 mL
Pure water (MilliQ or distilled)NANA490 mL
AddAmount490 mL water to sterile bottle
AddAmount300 mL ofConcentration5 Molarity (M) NaCl
AddAmount200 mL ofConcentration100 % volume Ethanol
AddAmount10 mL ofConcentration1 Molarity (M) Ph7.2 TRIS
Agitate to fully mix buffer solution
4S-WB2 composition:
ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer
NaCl5 M100 mM20mL
Ethanol100%80%800mL
TRIS pH 7.21 M10 mM10mL
Pure water (MilliQ or distilled)NANA170mL
AddAmount170 mL water to sterile bottle
AddAmount20 mL ofConcentration5 Molarity (M) NaCl
AddAmount800 mL ofConcentration100 % volume Ethanol
AddAmount10 mL ofConcentration1 Molarity (M) Ph7.2 TRIS
Agitate to fully mix buffer solution
Prepare two tubes containing lysis salts (one for the sample and one as a matched negative control) by addingAmount9.5 g of sodium chloride to each sterile 50mL tubes.
MakePh7.2 TE buffer (Concentration1 Molarity (M) TRIS,Concentration100 millimolar (mM) EDTA).
AddAmount400 µL of TE buffer to each 50mL tube with salt. Gently shake.
Sample preparation, RNA preservation and particle lysis
Sample preparation, RNA preservation and particle lysis
Obtain aAmount40 mL wastewater sample and pour directly into the pre-salted tube. Agitate sample until all NaCl dissolves in the wastewater. Maintain atTemperature4 °C during transport to the lab.
Raw wastewater containing NaCl, TRIS & EDTA. With the salt and the wastewater, the total volume in the tube will be about 44mL.
Note
Here, NaCl lyses lipid-protein envelopes, denatures proteins and disrupts RNA-protein interactions. EDTA inhibits the enzymatic degradation of RNA by RNases present in wastewater and TRIS provides optimal buffering conditions for nucleic acids.



Obtain Amount40 mL sterile 1x PBS and pour directly into the second pre-salted tube. Agitate sample until all NaCl dissolves in the PBS. Maintain atTemperature4 °C during transport to the lab. Perform same steps with the PBS-only negative control as described below for the wastewater sample.
Resuspend dry bovine coronavirus stock (Bovilis Coronavirus Calf Vaccine) inAmount2 mL of PBS. Dilute this resuspended stock into PBS at a dilution of 1:10 (Amount100 µL of stock into Amount900 µL PBS). Spike 50uL of diluted bCoV into the wastewater sample as a recovery efficiency control. Agitate sample to fully mix bCoV or other spiked-in controls with the wastewater sample.


ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445

Note
Other recovery controls can be used instead of bCoV, such as Phi6 bacteriophage. In addition, purified RNAs can be used to quantify the extraction efficiency of "free RNA".


Heat Amount200 µL of remaining bCoV spike-in aliquot atTemperature75 °C forDuration00:30:00 . Freeze for later quantification via RT-qPCR. This enables more accurate assessment of the bCoV spike-in concentration.
30m
(OPTIONAL) Heat inactivate sample atTemperature70 °C forDuration00:45:00 . Our unpublished analyses have shown that this step may slightly improve some RNA species' enrichment and detection.

45m
Filter the sample through a 5-um PVDF filter via syringe filtration or funnel top vacuum into a sterile 100mL tube.

Syringe filter setup: Wastewater is filtered through a 47-mm reusable filter membrane holder.
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700

ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700

ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
Wastewater filtering through a 5-um PVDF filter in a Pall filter holder.


Direct RNA extraction (RNA Binding, Washing, Eluting)
Direct RNA extraction (RNA Binding, Washing, Eluting)
Add Amount40 mL ofConcentration70 % volume to theAmount40 mL of filtrate.

Filtered sample before ethanol addition. Filtrate should be semi-clear.

Agitate sample to mix ethanol and wastewater lysate.
Attach Zymo III-P (or other) silica spin column to a vacuum manifold. Agitate the wastewater and lysate by inverting the tube five times, then pour into the spin column and vacuum the fullAmount80 mL of wastewater lysate & ethanol through the spin column.
Note
Commercial silica spin columns vary in their silica membrane packing tightness, changing the flow rate of lysed wastewater. We advise the use of the Zymo III-P column to avoid column clogging issues, but columns such as the Qiagen RNeasy, QIAamp Mini Spin and Zymogen II-CR can act as substitutes, depending on vacuum strength and sample particulate content. Large-format "maxiprep" style columns are also able to purify wastewater RNA, but require a large volume RNA elution up to 20mL (Step 13) and a dowstream precipitation-concentration step (Isopropanol precipitation, see companion protocol, Step 12) .
Passing lysed & filtered samples through Zymo III-P columns for direct RNA capture.
ReagentEZ-Vac Vacuum ManifoldZymo ResearchCatalog #S7000

ReagentZymo III-P columnZymo ResearchCatalog #C1040-5

VacuumAmount5 mL wash buffer #1 (4S-WB1) through the silica spin column.
VacuumAmount10 mL wash buffer #2 (4S-WB2) through the silica spin column.




RNA elution
RNA elution
Detach silica spin column from vacuum manifold, remove any attached reservoirs/funnels and place column into a 1.5-mL centrifugation-compatible flowthrough collection tube.
Centrifuge silica spin column in tube atCentrifigation10000 x g, 4°C, 00:02:00 to remove any residual 4S-WB2 present in the column.

Discard the collection tube and place silica column into a new 1.5-mL centrifugation-compatible flowthrough collection tube.
Pre-warmAmount200 µL of ZymoPURE elution buffer or Amount200 µL Ph8 TE buffer per RNA sample toTemperature50 °C in a heat block, waterbath or incubator.
ReagentZymoPURE Elution BufferZymo ResearchCatalog #D4200-7-30

ReagentTE buffer

AddAmount200 µL of pre-warmed elution buffer to each silica spin column. Incubate the elution buffer and column + collection tube assembly in a heat block or incubator warmed toTemperature50 °C forDuration00:10:00 .

Spin atCentrifigation10000 x g, 37°C, 00:05:00 to elute RNA from the column.
The flowthrough present in the collection tube contains the purified RNA.

Storage
Storage
The eluted RNA is now ready for downstream analysis. Store RNA atTemperature4 °C for same-day use or freeze atTemperature-80 °C for later use and storage.