Aug 06, 2020

Public workspaceExtraction of RNA from Wastewater Primary Solids Using a Direct Extraction Method for Downstream SARS-CoV-2 RNA Quantification

DOI
dx.doi.org/10.17504/protocols.io.bi6skhee
  • 1Stanford University;
  • 2University of Michigan
  • Coronavirus Method Development Community
  • Wastewater-based epidemiology working group
Alexandria B Boehm
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DOI: dx.doi.org/10.17504/protocols.io.bi6skhee
Protocol Citation: Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, Alexandria B Boehm 2020. Extraction of RNA from Wastewater Primary Solids Using a Direct Extraction Method for Downstream SARS-CoV-2 RNA Quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.bi6skhee
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 have used this protocol extensively with solids from a number of wastewater treatment plants.
Created: July 30, 2020
Last Modified: August 06, 2020
Protocol Integer ID: 39858
Keywords: wastewater, solids, SARS-CoV-2, influent, coronavirus, virus,
Disclaimer
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Abstract

Overview

This SOP describes pre-analytical procedures to be followed for the isolation and identification of SARS-CoV-2 RNA in primary settled solids samples from wastewater treatment plants. This protocal should be paired with our digital PCR protocal.


The protocol follows the approximate workflow:
  1. Separate and aliquot sample (~4 hrs)
  2. Spike with BCoV (~1 hr)
  3. Extract RNA (~ 12 hours total, may be split into 2-3 steps/days)
  4. Inhibitor removal (~1 hr)

Biosafety Concerns

Concentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines.

This protocol was developed by Stephanie Loeb, Katy Graham, Marlene Wolfe, Krista Wigginton, and Alexandria Boehm at Stanford University and University of Michigan.
Materials
MATERIALS
ReagentPhenol-chloroform-isoamyl alcohol 25:24:1 (PCI)Invitrogen - Thermo FisherCatalog #15593049
ReagentRNeasy® PowerSoil® Total RNA Kit QiagenCatalog #12866-25
ReagentOneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
STEP MATERIALS
ReagentRNeasy® PowerSoil® Total RNA Kit QiagenCatalog #12866-25
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo FisherCatalog #15593031
ReagentOneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030

Separate Solids and Spike with BCoV


Materials

  • 100p pipet tips
  • 50mL falcon tubes
  • Percent solids materials (listed in part 2)
  • PowerBead Tubes (from RNeasy PowerSoil Kit)
  • 5 mL cryovials
  • Bovine coronavirus (BCoV) stock
  • 25mL serological pipettes
  • Disposable polypropylene spatulas


Equipment

  • Biosafety cabinet
  • 100p pipet
  • Centrifuge with high speed rotor
  • Balance
  • Tube rack
  • Automatic serological pipettor



Percent Solids Measurements

Materials

  • Aluminum weigh dish
  • Disposable polypropylene spatulas
  • Aluminum foil

Equipment

  • Oven set at at 105°C
  • Analytical balance
  • Dehydrator (not essential)



RNA Extraction


Materials:

  • Qiagen RNeasy PowerSoil Total RNA extraction kit
  • LoBind tubes (for -80°C storage)
  • Molecular grade mixture of 25 parts phenol, 24 parts chloroform, and one part isoamyl alcohol
  • 1000p pipet tips (RNAse/DNase-free)
  • 200p pipet tips (RNAse/DNase-free)
  • 10p pipet tips (RNAse/DNase-free)
  • Disposable polypropylene spatulas


Equipment:

  • 1000p pipet
  • 200p pipet
  • Heat block (in BSC)
  • 10p pipet
  • Vortex with 15 mL falcon tube adapter (in BSC)
  • Centrifuge



Inhibitor Removal


Materials

  • Zymo OneStep PCR Inhibitor Removal Columns
  • LoBind 1.5 mL tubes
  • P100 pipet tips- molecular biology grade


Equipment

  • Mini-centrifuge
  • 100p pipet
Protocol materials
ReagentRNeasy® PowerSoil® Total RNA Kit QiagenCatalog #12866-25
ReagentOneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentRNeasy® PowerSoil® Total RNA Kit QiagenCatalog #12866-25
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo FisherCatalog #15593031
ReagentOneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030
ReagentPhenol-chloroform-isoamyl alcohol 25:24:1 (PCI)Invitrogen - Thermo FisherCatalog #15593049
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentOneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030
ReagentRNeasy® PowerSoil® Total RNA Kit QiagenCatalog #12866-25
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo FisherCatalog #15593031
Safety warnings
Concentration and extraction procedures that utilize raw samples must adhere to strict Biosafety Level 2+ procedures. These procedures should be performed in a dedicated room. Downstream products may be handled using standard laboratory safety guidelines.

Before Starting
Before Starting
30m
30m
Day before sample processing:

  • Thaw sample DurationOvernight at Temperature4 °C . Record the weight and volume of the sample.

Day of sample processing:

  • Place aluminum dishes in oven Duration01:00:00 before use
  • Set centrifuge temperature to Temperature4 °C Duration00:30:00 before use



Separate Solids and Spike BCoV
Separate Solids and Spike BCoV
5h
5h
Load Amount40 mL samples of primary sludge at a time into the centrifuge rotor. Balance with equally weighted tubes filled with water.

Centrifuge at Centrifigation14500 rpm, 4°C, 00:30:00 , (equivalent to 24,000 xg) .

Safety information
Bring the centrifuge rotor to the BSC before loading or opening.


Decant the supernatant into an empty 50 mL falcon tube or larger container. Record the weight and volume of the remaining solids.
Safety information
Discard liquid as biohazard waste.
Separated solids with liquid removed

Transfer solids for percent solids measurement: Transfer a generous (~Amount500 mg ) aliquot of the solid fraction onto a pre-weighed aluminum weigh dish. The aluminum weigh dish should be used directly out of the oven and not allowed to rehydrate. Record the total weight of the weigh dish before covering with aluminum and transferring to the oven to measure dry weight following EPA method Method 106.3. (Page 62) Link to EPA method. See page 62.
Note
If there is less than 2 g of solids available, % solids may be performed with a smaller aliquot.


Aliquot solids as follows into tarred or pre-weighed tubes using a sterile spatula. Note exact mass aliquoted, with a target range from Amount2.0 g to Amount2.5 g .

One 2g aliquot can be place directly into a 15 mL PowerBead tubes from RNeasy PowerSoil Total RNA extraction kit to prepare for extraction. Depending on the number of replicates required, additional aliquots can also be placed in PowerBead tubes for extraction.
ReagentRNeasy® PowerSoil® Total RNA Kit QiagenCatalog #12866-25

Additional 2g aliquots may be placed in as many 5mL cryovials as possible for storage at Temperature-80 °C .

Individually spike each sample that has been aliquoted in a 15mL PowerBead tube with 20 µL BCoV stock. Pipet the spike directly into the solids and mix well with a spatula to homogenize. Equilibrate the sample at 4°C for 1 hour.
Note
Vials of BCoV vaccine should be rehydrated with molecular grade water before use via needle and syringe, and the solution may be stored at 4°C. For each batch of BCoV, a known dilution in PBS should be processed and analyzed in order to determine the concentration spiked

ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445

Store samples in PowerBead tubes at Temperature-80 °C until extraction. Archive samples in cryovials at Temperature-80 °C .

Extract RNA
Extract RNA
12h
12h
Prepare for extractions using the RNeasy PowerSoil Total RNA kit to extract and isolate viral RNA into 100uL of eluent. This extract can be used for one step ddPCR or reverse transcribed for qPCR.


Before starting
  • Prepare a sterile workspace
  • Heat Solution IRS at Temperature60 °C until any precipitate dissolves.
  • Wear RNase-free gloves at all times and remove RNase from the work area.
Safety information
All sample processing must occur in the biosafety cabinet until the bead beating step is completed. After bead beating, cells have been completely lysed and the samples are considered non-infectious.


Remove samples from the freezer and place TemperatureOn ice . Samples do not need to be thawed before processing. Use Solution IRS while it is still warm.

For processing samples already in PowerBead tube

Add Amount2.5 mL of PowerBead Solution,Amount0.25 mL of Solution SR1 and Amount0.8 mL of Solution IRS directly to the tube containing the sample. Vortex the tube vigorously to suspend the solids.
Note
If the sample is not in a powerbead tube, perform step 11 in the sample tube, transferring the entire mixture to the PowerBead tube prior to step 12. If a large amount of residual solids remain in the tube after step 11, perform step 4 in two “washes” of 1.75 mL each phenol/chloroform/isoamyl alcohol. If a disposable spatula was used, the spatula can also be rinsed in this solution as well.

Add Amount3.5 mL of phenol/chlorogorm/isoamyl alcohol (pH 6.5–8.0, not included in kit) to the sample tube. Vortex vigorously.
ReagentUltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)Thermo FisherCatalog #15593031

Cap and vortex the PowerBead Tube to mix until the biphasic layer disappears. Place the PowerBead Tube on a Vortex Adapter and vortex at maximum speed for Duration00:15:00 .

Remove the PowerBead Tube and centrifuge at Centrifigation2500 x g for Duration00:10:00 at TemperatureRoom temperature . After centrifugation, two or three phases may be visible. Total nucleic acids are retained in the upper aqueous phase.

Transfer the upper aqueous phase  to a clean 15 ml Collection Tube.  Take care not to transfer material from the lower phases.

Safety information
After transferring the upper aqueous, the remaining fractions can be discarded as BSL2 waste.

Add Amount1.5 mL of Solution SR3 to the aqueous phase and vortex to mix. Incubate at Temperature4 °C for Duration00:10:00 and then centrifuge at Centrifigation2.500 x g for Duration00:10:00 at TemperatureRoom temperature .

Transfer the supernatant, without disturbing the pellet, to a new 15 ml Collection Tube.
Add Amount5 mL of Solution SR4 to the supernatant in the Collection Tube and invert or vortex to mix.
Incubate at Temperature-20 °C for Duration00:30:00 .


Note
Samples can also be incubated at Temperature-20 °C DurationOvernight , to continue procedure on the next day.



Centrifuge at Centrifigation2500 x g for Duration00:30:00 .

Decant the supernatant and invert the 15 ml Collection Tube on a paper towel for Duration00:05:00 .
Note
Check that the pellet seems to be firmly in the bottom of the tube - if so, you can carefully pour off the supernatant to decant.


Shake Solution SR5 to mix and add Amount1 mL to the 15 ml Collection Tube. Resuspend the pellet completely by repeatedly pipetting or vortexing.


Note
The pellet will be difficult to resuspend. Place the tube in a heat block or water bath at Temperature45 °C for Duration00:10:00 , followed by vortexing and/or pipetting up and down. Repeat until the pellet is resuspended. When the pellet is fully re-suspended, the solution will appear as a relatively opaque homogenous cloudy yellow-white solution. Continue heating and vortexing until no visible precipitates remain. Failing to fully re-suspend the pellet will result in large downstream losses of nucleic acids.

Prepare one JetStar Mini Column from the RNeasy kit for each RNA isolation sample. Remove the cap of a 15 ml Collection Tube (provided) and place the JetStar Mini Column inside it. The column will hang in the Collection Tube.
Add Amount2 mL of Solution SR5 to the JetStar Mini Column. Allow it to completely gravity flow through the column and collect in the 15 ml Collection Tube.
Note
Do not allow the column to dry out before loading sample in the next step.

Add the RNA isolation sample from Step 21 onto the JetStar Mini Column and allow it to gravity flow through the column into the 15 ml Collection Tube.

Note
Expect the flow-rate through the column to be slow (and highly variable between samples) - reserve enough time accordingly. If possible, allow samples to flow through under gravity only. If time to flow exceeds 1 hr, add positive pressure to the column using a syringe plunger. Do not force the flow at a rate of >1 drop per second.

Add Amount1 mL of Solution SR5 to the JetStar Mini Column and allow it to completely gravity flow into the 15 ml Collection Tube.
Note
Apply positive pressure with syringe plunger to samples that are stalled. Do not force the flow at a rate of >1 drop per second.


Transfer the JetStar Mini Column to a new 15 ml Collection Tube. Shake Solution SR6 to mix and then add Amount1 mL to the JetStar Mini Column to elute the bound RNA. Allow Solution SR6 to gravity flow into the 15 ml Collection Tube.

Note
Apply positive pressure with syringe plunger to samples that are stalled. Do not force the flow at a rate of >1 drop per second.


Transfer the eluted RNA to a 2.2 ml Collection Tube. Add Amount1 mL of Solution SR4. Invert at least once to mix and incubate at Temperature20 °C for a minimum of Duration00:10:00 .

Note
Samples can also be incubated at Temperature-20 °C DurationOvernight , to split continue the procedure on the next day.

Centrifuge the 2.2 ml Collection Tube at Centrifigation13000 x g for Duration00:15:00 to pellet the RNA.

Decant the supernatant and invert the 2.2 ml Collection Tube onto a paper towel for Duration00:30:00 to air dry the pellet.

Resuspend the RNA pellet in Amount100 µL of Solution SR7. Observe the pellet carefully to make sure it is fully re-suspended. The RNA is now ready for downstream applications.

Note
Observe the base of the tube while slowly rotating the sample - there should not appear to be any material adhered to the side or base of the tube, and the solution should flow smoothly.


Inhibitor Removal
Inhibitor Removal
1h
1h
Use the Zymo OneStep PCR Inhibitor Removal Columns to remove inhibitors that were co-concentrated with the nucleic acids from the extracts to prevent downstream issues.

ReagentOneStep PCR Inhibitor Removal KitZymo ResearchCatalog #D6030

Place one column into one collection tube for each sample you need and add Amount600 µL of Prep solution to each column.

Centrifuge collection tubes in columns at Centrifigation8000 x g for Duration00:03:00 .

Discard collection tube with flow through and load column into a labeled 1.5mL LoBind tube.
Add the full volume of RNA/DNA extract (Amount100 µL ) to each column.

Centrifuge LoBind tubes with column at Centrifigation16000 x g for Duration00:03:00

This extract can be used for cDNA synthesis or as template for a one-step PCR reaction. Immediately perfom Nanodrop analysis, Qubit analysis, and/or reverse transcriptase if desired, or store eluent at Temperature-80 °C for later use.