Dec 14, 2022
Membrane Filtration for SARS-CoV-2 Viral Capture
- Caleb Centrell1,
- Jamie VanTassell1,
- Julia Raymond1,
- Marlene K Wolfe1,
- Pengbo1,
- Christine Moe1,
- Pengbo Liu1
- 1Center for Global Safe WASH, Rollins School of Public Health, Emory University, Atlanta Georgia USA
Protocol Citation: Caleb Centrell, Jamie VanTassell, Julia Raymond, Marlene K Wolfe, Pengbo, Christine Moe, Pengbo Liu 2022. Membrane Filtration for SARS-CoV-2 Viral Capture . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmno85g3p/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: December 10, 2021
Last Modified: December 14, 2022
Protocol Integer ID: 55836
Keywords: membrane filtration, SARS-CoV-2, COVID-19, wastewater
Funders Acknowledgement:
Ceres Nanosciences – NIH RADx Tech
Grant ID: 75N92021C00012
Abstract
This protocol describes how to perform membrane filtration vacuum technology with wastewater grab samples to allow for SARS-CoV-2 RNA extraction.
Image Attribution
shutterstock.com
Materials
Equipment:
- Sterilized forceps
- Alcohol lamp burner
- Benchtop scale
- Centrifuge (Thermo Scientific. SORVALL RC 6+)
- pH Probe ( Fisher Scientific. Fisherbrand accumet AB15 plus. pH meter CAT# 13-620-631)
- 5% HCl solution
- 5% NaOH solution
- Aluminum Foil
- 10% Bleach solution
- 70% Ethanol solution
- 100% Ethanol (96% is what is used in the lab)
- Concentrated bleach
- Benchtop Protector Paper
- Eppendorf Research Plus Single Channel Pipette
- LabGard Biological Safety Cabinet - Class 2 A2 Biosafety Cabinet
- Autoclave
- Benchtop biohazard bag(s)
- Benchtop biohazard bag stand ( GENESEE SCIENTIFIC CORPORATION. CAT# 30-164)
- Automatic handheld repeater (EppendorfCAT#13-683-552)
Materials Needed (per Sample):
- (1) Field Sample collected and stored at 4C until processing
- (1) Autoclaved centrifuge bottle and lid with O-ring
- (1) Autoclaved 500mL beaker
- (1) Autoclaved Membrane Filtration Cup contained within a (Fisher Scientific.CAT#01-812-55) Sealed Bag
- (1) Sterile 0.45μm (Millipore. CAT#HAWP04700) membrane filter paper
- (1) Powdered Magnesium Chloride (MgCl2)
- (1) 104 10μl Bovine Respiratory Syncytial Virus (BRSV) processing control aliquot
- (1) 25mL graduated pipette
- (1) Qiagen RNeasy Mini Kit (Cat. No. 74106)
Protocol materials
Buffer RLTQiagenCatalog #79216
In 3 steps
Buffer RW1QiagenCatalog #1053394
Step 49
Buffer RPEQiagenCatalog #1018013
In 2 steps
Before start
Biosafety Level 2 (BSL-2) certification is required due to the nature of the target pathogen.
Sterilize Biological Hood
Sterilize Biological Hood
Turn on the biological hood vent and wait for airflow to start circulating.
Carefully remove the vacuum flask and hoses that are attached to the vacuum vent within the biological hood and dispose of any liquid present in the correct biological hazard trash container.
Wash and rinse the vacuum hoses using a mixture of hot water and concentrated bleach at a 2:1 ratio.
Pour concentrated bleach solution into the vacuum flask to cover the entire bottom surface.
Spray 10% bleach solution inside the biological hood and wipe all surfaces with paper towels.
After spraying the 10% bleach solution, spray 70% ethanol solution inside the biological hood and wipe all surfaces with paper towels.
Carefully reattach the vacuum flask and vacuum hoses to the vacuum vent within the biological hood.
Place two pieces of benchtop surface paper underneath the membrane filtration vacuum apparatus that is inside of the sterilized biological hood.
Sterilize Forceps
Sterilize Forceps
Pour 100% ethanol into a smaller autoclaved beaker to a level that allows for the entire end of forceps to be submerged.
Carefully light a alcohol lamp burner.
Place the forceps that have been submerged in the 100% ethanol and burn the ethanol off to create a visible flame.
Place the now sterilized forceps into the biological hood in a position so that the sterilized end is not touching a physical surface to remain aseptic.
When forceps are used or come into contact with any surface or filter paper that is contaminated with a sample, they must be re-sterilized using the method given above.
Preparation
Preparation
Prepare and collect all supplies necessary for the number of samples that will be concentrated using membrane filtration.
Note
- All glassware, membrane filtration cups, and centrifuge bottles should be autoclaved with aluminum foil covering any opening
- Place all autoclaved and sterilized materials underneath the sterilized biological hood
- Place a biohazard benchtop waste bag into the biological hood on a stand for easy disposal of any contaminated material used during the procedure
Sample Processing Procedure
Sample Processing Procedure
5m
5m
Remove the grab sample from the 4°C refrigerator and place under the sterilized biological hood.
Aseptically pour 300 mL of the grab sample into an autoclaved 500mL beaker that is labeled with a sample identifier using labeling tape.
After 300mL of the grab sample has been measured, aseptically pour the aliquoted sample into an autoclaved centrifuge bottle that is labeled with the sample identifier using labeling tape.
Note
Using sterilized forceps, ensure that the O-ring of the autoclaved centrifuge bottle is securely inside of the centrifuge bottle lid.
Using aseptic technique, secure the centrifuged bottle lid onto the centrifuge bottle containing the sample and secure the lid by turning a quarter of the way past tight.
Balance the sample centrifuge bottles using a benchtop scale that has been sterilized using 10% bleach solution followed by 70% ethanol solution.
Note
- The benchtop scale should be placed within the biological hood to ensure no sample contamination outside of the biological hood area.
- Measure each sample and centrifuge bottle with the lid attached and record the weight.
- Using DI water, adjust the weight of each sample centrifuge bottle to be within 0.5 grams of each other.
Centrifuge the sample(s) for 5000 rpm, 00:05:00 .
Equipment
Sorvall™ RC 6 Plus
NAME
Centrifuge
TYPE
Thermo Scientific™
BRAND
12121680
SKU
LINK
Note
Ensure that the centrifuge is balanced and running properly according to manufacturer specifications.
5m
Remove the centrifuged sample(s) from the centrifuge and carefully place them back under the biological hood.
Note
Take care to not disturb any pellet that has formed at the bottom of the centrifuge bottle.
Measure out 0.71 g +/- 0.02g of MgCl2 using benchtop scale paper and an autoclaved chemical spatula.
Pour the measured MgCl2 into the 500mL beaker that was initially used to measure the sample volume. Put a magnetic stir bar into the beaker and stir the sample on the stirrer for 00:15:00 .
15m
Aseptically pour the centrifuged sample into the identically labeled 500mL beaker that now has the MgCl2 to create a final concentration of 25mM.
Note
Ensure while pouring liquid samples to not pour any pellet or solids into the beaker along with the liquid.
Place a benchtop calibrated pH probe underneath the biological hood and sterilize the probe, excluding the glass measurement part, and workspace with 10% bleach solution followed by 70% ethanol solution.
Adjust the pH to 3.5 +/- 0.1 using 5% HCl and 5% NaOH solutions.
Note
Use individual pipette droppers for both the 5% HCl and 5% NaOH solutions (do not mix the pipettes).
Use a Eppendorf Research Plus Single Channel Pipette to add 10 µL of BRSV aliquot into the sample.
Note
- Gently pipette to the second stop to expel all 10µL of BRSV into the sample.
- Use a new pipette tip for each sample.
Cover the opening of the sample beakers with aluminum foil and secure the foil by either wrapping around the edge of the opening or with autoclave tape.
Gently and carefully swirl the sample to allow for homogeneous mixing of the BRSV throughout the sample beaker.
Place the sample beaker into a 4 °C refrigerator for 00:30:00 .
30m
While the sample sits in the refrigerator for 30 minutes, place an autoclaved membrane filtration cup into each of the vacuum manifold openings and secure with a quarter turn to create a seal.
Without disturbing the lower half portion of the membrane filtration cup, remove the top cup portion and place topside down onto the benchtop paper that was placed at the start of the procedure preparation.
Using the sterilized forceps, aseptically place a 0.45µM membrane filter paper onto the opening of the membrane filtration cup that is still attached to the vacuum manifold.
When initially setting up the vacuum membrane filtration manifold with filter papers, sterilization of forceps can be done a singular time when all membrane filter cups are autoclaved and placed into the biological hood on the vacuum manifold with no sample(s) present.
After all membrane filtration cups have a 0.45µM membrane filter paper placed onto their respective opening, reattach the top of the membrane filtration cups to their base.
Re-sterilize the forceps and place into an aseptic position for later use after sample processing.
Label the membrane filtration cups with their respective sample identifier.
Note
Label on the individual membrane filtration cup using labeling tape or label the benchtop paper that is directly in front of the membrane filtration cup.
After the 30-minute refrigeration period is over, pour 150 mL of the sample into a membrane filtration cup that has the membrane filter paper and is sealed tightly on the vacuum manifold.
Note
Place the remaining 150mL of sample within its beaker back into the 4 °C refrigerator while the sample is being processed through the vacuum manifold.
Check the seals of the vacuum hoses and vacuum flask to make sure that the seals are secure to prevent leakage of any sample liquid.
Turn the vacuum vent that is attached to the biological hood on to begin the vacuum process, and subsequently turn the valve on the vacuum manifold for each sample that is being processed.
Check the seal on the vacuum hoses and flask once again after the vacuum process has begun.
Throughout the filtration process, frequently check the flow of sample liquid in the membrane filtration cups until the whole sample volume has filtered through the membrane. After 30-45 minutes, if the sample is draining very slowly and a large amount of sample volume still remains to be filtered, follow sub-steps 39.1 - 39.12.
Note
- The margin of error of sample volume should be no more than 10% (15mL).
- Sample turbidity can affect the speed of flow through and can also cause filter paper to clog, which inhibits the flow rate tremendously.
Retrieve the sample beaker from the 4°C and place under the biological hood.
Retrieve a 25mL graduate pipette and an automatic repeater pipette.
Turn the vacuum valve off for the respective sample.
Pipette the sample that is remaining within the membrane filtration cup back into the sample beaker and measure the volume amount that is removed.
After the sample is removed from the membrane filtration cup, remove the top half of the membrane filtration cup and place top down onto the benchtop paper.
Using sterilized forceps, roll the membrane filter paper that contains the filtered sample and place into a labeled DNA LoBind tube for RNA extraction.
Note
- Rolling of the membrane filter paper should be done with the sample-containing side exposed outwards to the walls of the DNA LoBind tube (i.e., roll the filter so that the dirty side is facing outwards). This improves release of the viral particles from the filter when the tube is shaken later on.
- Always sterilize forceps in between each use.
Using an Eppendorf Research Plus Single Channel Pipette, place 400 µL of Buffer RLTQiagenCatalog #79216 from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the labeled sample DNA LoBind tube that contains the first sample membrane filter paper.
Note
The total volume of Buffer RLT will be 800 µL. However, the tube will overflow if it is all added at once. Therefore, only 400 µL is aliquoted to submerge the first filter paper (to prevent it from drying out) while the second paper filters the remaining sample volume in the membrane filtration cup. After the second filter is added to the tube, aliquot the remaining 400 µL of Buffer RLT.
Place the sample DNA LoBind tube into a 4℃ refrigerator while the remaining sample volume is filtered through a secondary membrane filter paper.
Re-sterilize the forceps and aseptically place a new 0.45 µM membrane filter paper onto the membrane filtration cup opening and reseal using the top half of the membrane filtration cup.
Using the same 25mL graduated pipette, replace the measured sample volume into the cup and turn the vacuum valve back to the open position.
Once the remaining sample has filtered through the membrane, remove the top half of the cup, roll the membrane filter paper that contains the filtered sample, and place into the labeled DNA LoBind tube that contains the first membrane.
Note
The DNA LoBind tube now contains two filters for the same sample.
Add the remaining 400 µL ofBuffer RLTQiagenCatalog #79216 to the DNA LoBind tube to create a total volume of 800 µL.
Once the full sample volume (150 mL +/- 15 mL) has filtered through the membrane, remove the top half of the membrane filtration cup and place top down onto the benchtop paper.
Using sterilized forceps, roll the membrane filter paper that contains the filtered sample and place into a labeled DNA LoBind tube for RNA extraction.
Note
- Rolling of the membrane filter paper should be done with the sample-containing side exposed outwards to the walls of the DNA LoBind tube (i.e., roll the filter so that the dirty side is facing outwards). This improves release of the viral particles from the filter when the tube is shaken later on.
- Always sterilize forceps in between each use.
- Using an Eppendorf Research Plus Single Channel Pipette, place 800 µL of Buffer RLTQiagenCatalog #79216 from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the labeled sample DNA LoBind tube that contains the membrane filter paper.
Store DNA LoBind tubes with filters and Buffer RLT in a 4℃ refrigerator until all samples are processed and ready for RNA extraction.
RNA Extraction
RNA Extraction
Insert DNA LoBind tubes that contain the sample filters into a vortex adapter and vortex for 00:10:00 .
10m
Centrifuge tube for 00:03:00 at maximal speed.
3m
Pipette the available liquid into a new, labeled tube. You may discard the tube with the filter(s) afterwards.
Note
Using the tip on the pipette, push the filter aside to extract as much sample volume as possible. Repeat this 2-3 times, aiming to collect approximately 700 µL of sample liquid.
Aliquot an equal volume of 70% molecular ethanol to the sample. Mix well by pipetting the mixture up and down several times. Do not vortex after this step.
Note
- For example, if you extracted 700 µL of sample fluid from the tube with the filters, than you would add 700 µL of 70% molecular ethanol.
Transfer 700 µL of the sample mixture into a labeled RNeasy spin column. Centrifuge for 00:00:30 at full speed. Discard filtrate. Repeat until all of the sample is filtered through the spin column.
30s
Aliquot 700 µL of Buffer RW1QiagenCatalog #1053394 to the RNeasy spin column. Centrifuge for 00:00:30 at full speed. Discard filtrate.
30s
Aliquot 500 µL of Buffer RPEQiagenCatalog #1018013 to the RNeasy spin column. Centrifuge for 00:00:30 at full speed. Discard filtrate.
30s
Aliquot 500 µL of Buffer RPEQiagenCatalog #1018013 to the RNeasy spin column. Centrifuge for 00:02:00 at full speed. Discard filtrate.
2m
Transfer spin column to new 2 mL collection tube and centrifuge for 00:01:00 at full speed. Discard collection tube and filtrate.
1m
Place the RNeasy spin column into a labeled 1.7 mL microcentrifuge tube. Add 50 µL of RNA-free water to the center of the spin column. Incubate for approximately 00:01:00 at room temperature.
Note
This microcentrifuge tube will contain the final RNA for PCR. Ensure that the label will be able to withstand storage in a freezer until sample is ready for PCR or for archiving purposes.
1m
Centrifuge for 00:01:00 at full speed.
1m
Add another 50 µL of RNA-free water to the center of the spin column. Incubate for approximately 00:01:00 at room temperature.
1m
Centrifuge for 00:01:00 at full speed.
1m
Close the cap of the 1.7 mL tube containing the final RNA (you will have a final volume of 100 µL ).
Store RNA at -20 °C until it is ready for PCR.
Post-Sample Processing Clean-Up Procedure
Post-Sample Processing Clean-Up Procedure
Place all benchtop paper and all disposable materials into the benchtop biohazard bag.
Place the benchtop biohazard bag into the correct biohazard floor trash can for future autoclaving.
Sterilize forceps that were used and place into their respective areas for future use.
Place all contaminated membrane filtration cups, sample beakers, centrifuge bottles, and any other glassware into a sink for cleaning prior to autoclaving.
Using concentrated bleach, adequately wash all glassware, centrifuge bottles, and membrane filtration cups and scrub any solids or residue off.
Using lab-grade soap and hot water, adequately wash all glassware, centrifuge bottles, and membrane filtration cups before placing on a drying rack.
Follow current lab procedures for autoclaving and store autoclaved materials for future membrane filtration processing of samples.
Spray 10% bleach solution throughout the biological hood and wipe all surfaces, manifold, and potentially contaminated surfaces with paper towels.
Spray 70% ethanol solution throughout the biological hood and wipe all surfaces, manifold, and potentially contaminated surfaces with paper towels.
Close the biological hood and turn off all vents and lights.