Jan 21, 2022
Skim Milk Flocculation and RNA Extraction for SARS-CoV-2 Viral Capture
- Jillian Dunbar1,
- Stephen P Hilton1,
- Jamie VanTassell1,
- Julia Raymond1,
- Marlene K Wolfe1,
- Pengbo1,
- Christine Moe1
- 1Center for Global Safe WASH, Rollins School of Public Health, Emory University, Atlanta GA USA
Protocol Citation: Jillian Dunbar, Stephen P Hilton, Jamie VanTassell, Julia Raymond, Marlene K Wolfe, Pengbo, Christine Moe 2022. Skim Milk Flocculation and RNA Extraction for SARS-CoV-2 Viral Capture. protocols.io https://dx.doi.org/10.17504/protocols.io.b2uwqexe
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 14, 2021
Last Modified: January 21, 2022
Protocol Integer ID: 55926
Keywords: skim milk flocculation, wastewater, SARS-CoV-2, COVID-19, Moore swab
Funders Acknowledgements:
Rollins School of Public Health, Emory University
Grant ID: N/A
Abstract
This protocols describes the use of Skim Milk Flocculation to concentrate and extract SARS-CoV-2 from Moore swab wastewater samples. Included in this protocol are: materials and equipment, steps for processing Moore swabs, and steps for concentrating and extracting the virus using the Qiagen RNeasy Mini Kit.
Materials
Equipment:
- Stainless Steel Potato Ricer or Lab Paddle Stomacher (note: a Stomacher is more expensive but requires less effort for lab technician)
- Erlenmeyer flask
- pH Probe
- Centrifuge Tube
- 50 mL centrifuge tube
- 2 mL microcentrifuge tube
- 4in. by 4in. gauze
- Centrifuge
- Orbital Shaker
- RNeasy spin column
- Eppendorf Research Plus Single Channel Pipette
- LabGard Biological Safety Cabinet - Class 2 A2 Biosafety Cabinet
- Autoclave
Materials:
- Elution solution (contains: NaPP, Tween 80, Antifoam A, 10X PBS)
- Skim milk powder
- 5% HCl
- 5% NaOH
- 104 Bovine Respiratory Syncytial Virus (BRSV) per sample
- 70% molecular ethanol
- Qiagen RNeasy Mini Kit (Cat. No. 74106)
Protocol materials
Buffer RW1QiagenCatalog #1053394
Buffer RPEQiagenCatalog #1018013
Buffer RPEQiagenCatalog #1018013
RNAse-free Water
RNAse-free Water
Buffer RLTQiagenCatalog #79216
Buffer RLTQiagenCatalog #79216
Polyoxyethylene-80 (TWEEN 80)Bio Basic Inc.Catalog #TB0562.SIZE.500ml
double distilled water (ddH2O)
Skim Milk Powder
Moore Swab Processing
Moore Swab Processing
54m 30s
54m 30s
Two alternative methods are provided herein for initial swab processing: Lab Paddler Stomacher (Step 2) or a Potato Ricer (Step 3).
If using a Lab Paddler Stomacher:
Set the stomacher settings to paddle at speed 2 for 00:00:30 .
30s
Place Moore swab sample in a clean stomacher bag with 100 mL of Elution Solution (see Appendix I for preparation steps).
Place bag in stomacher so that the top of the bag is above the stomacher and close the handle. The stomacher will begin to paddle the swab for the programed time.
Fold the stomacher bag in half length-wise and pour the fluid into a labeled centrifuge bottle.
Note
- Squeeze the swab through the stomacher bag while pouring to release sample fluid contained in the swab.
- Be careful not to let the swab fall out of the bag while pouring the sample fluid into the centrifuge bottle.
Repeat Steps 2.1 - 2.4 until you have 300 mL of sample volume.
If using the potato ricer method:
Picture of stainless steel potato ricer used for squeezing wastewater fluid out of Moore swab.
Place Moore swab sample in a potato ricer and squeeze all the liquid out into a 600 mL beaker.
Note
- The first squeeze typically yields ~100mL of wastewater fluid.
- It is easier to squeeze the fluid into a beaker than a flask due to the wider mouth of the beaker.
Pour the squeezed liquid into a labeled 500 mL Erlenmeyer flask.
Note
- The 500 mL flask is used to measure the amount of sample volume (final volume will be approximately 300 mL).
- The beaker will be used to mix the swab with the Elution Solution. You will therefore need one flask and one beaker for each swab sample.
Place the Moore swab back in the beaker. Add 100 mL of Elution Solution (see Appendix I for preparation steps) into the beaker.
Gently knead the Moore swab 10 times using the pointed end of a clean 50 mL conical tube. Then, use the top/flat surface of the tube to knead the swab 10 more times.
Note
- Our lab team has found that the 50 mL conical tubes are a good tool for kneading the swabs; however, other instruments could be used to knead the swab.
- It is vital that the instrument should be either sterilized or replaced for each new sample to prevent cross-contamination.
Turn the swab over and repeat Step 3.4.
Put the swab back into the potato ricer, and squeeze all the liquid out into the beaker.
Pour the liquid into the 500 mL Erlenmeyer flask.
Repeat Steps 3.3 through 3.7 until there is 300 mL of sample liquid in the 500 mL Erlenmeyer flask.
Pour contents into a labeled centrifuge bottle.
Ensure all of the centrifuge bottles have similar masses (+/- 0.5g) so that the centrifuge is balanced.
Note
- Using a benchtop scale, weigh each centrifuge bottle (with the lid). Identify the heaviest sample, record its weight, and set it aside.
- Using DI water, adjust the weight of the remaining samples so that each bottle is within 0.5 g of each other.
Wipe down the benchtop scale after use using 10% bleach followed by 70% ethanol.
Centrifuge for 00:15:00 at 5000 rpm .
Note
- Utilize the full capacity of the centrifuge. For example, if your centrifuge can hold six 300 mL centrifuge bottles, prepare six samples for the centrifuge. While samples are centrifuging for 15 minutes, you can continue processing the remaining samples.
- If you have a number of samples that cannot be balanced, fill a centrifuge bottle with DI water to be within 0.5 grams of the other bottles and place in the centrifuge.
Centrifuge loaded at full capacity.
Balanced centrifuge.
15m
Pour only the supernatant back into the flask (should be approximately 250 mL ).
Note
- Take care not to let the aggregated solids fall into the flask as you are pouring the supernatant.
- Make sure you are using the same flask as the sample was originally processed in (flask should be labeled to prevent accidental cross-contamination).
Adjust the pH of the sample to 3.5 using 5% HCl and 5% NaOH using a pH probe.
Aliquot 10 µL of 104 Bovine Respiratory Syncytial Virus (BRSV) as a positive control to the sample. Mix well by pipetting the BRSV up and down 20 times in the sample before expelling it.
Note
Use a new tip for each sample.
Add 1 % volume of Skim Milk Solution (see Appendix II for preparation steps) to the sample volume.
Note
For example, if sample volume is 250 mL, add 2.5 mL of Skim Milk Solution.
Place aluminum foil over the opening of the flask.
Place sample on shaker 120 rpm, Room temperature , 02:00:00 .
Note
If a shaker is not available, place a sterilized magnetic stir bar in the sample and stir at room temperature at low speed for 2 hours.
RNA Extraction
RNA Extraction
54m 30s
54m 30s
Pour sample from flask into sterilized and labeled centrifuge bottle.
Centrifuge sample for 00:30:00 at 12000 rpm .
30m
Pour supernatant out (you will not use this liquid for RNA extraction).
Gently scrape off and discard a large portion of the pellet, leaving behind a thin film of pellet in the centrifuge bottle.
- Aliquot 800 µL of Buffer RLTQiagenCatalog #79216 from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the centrifuge bottle.
Mix well by pipetting the pellet and Buffer RLTQiagenCatalog #79216 up and down about 30 times or until the mixture looks homogeneous.
Pipette 800 µL of homogenized mixture into a labeled 2 mL DNA LoBind microcentrifuge tube.
Centrifuge tube for 00:03:00 at full speed.
3m
Transfer all of the sample into a new labeled tube.
Aliquot 800 µL of 70% molecular ethanol.
Note
- Mix well by pipetting the mixture up and down several times.
- Do not vortex after this step.
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
Add 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 filtrate and collection tube.
1m
Place the RNeasy spin column into a 1.7 mL labeled microcentrifuge tube. Aliquot 50 µL of RNAse-free Water (from RNeasy kit). Incubate for approximately 00:01:00 at room temperature.
Note
This labeled 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
Aliquot another 50 µL of RNAse-free Water into the the RNeasy spin column in the 1.7 mL labeled microcentrifuge tube. 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.
Appendix I - Prepare Elution Solution
Appendix I - Prepare Elution Solution
Prepare the Tween 80 stock by dissolving 1 mL of Polyoxyethylene-80 (TWEEN 80)Bio Basic Inc.Catalog #TB0562.SIZE.500ml in 100 mL of distilled water.
Prepare the Antifoam A stock by dissolving 1 mL of Antifoam A in 100 mL of distilled water.
Prepare the NaPP stock by dissolving 1 g of NaPP in 100 mL of distilled water.
Prepare the 10X PBS Solution
Mix together:
- 80 g NaCl
- 2 g KCl
- 14.4 g Na2HPO4
- 2.4 g KH2PO4
- 800 mL Ddwater
Adjust the solution to 7.4 by adding 5% NaOH and/or 5% HCl.
Note
- Swirl the mixture after adding either NaOH or HCl.
- Use a pH probe in between adding NaOH or HCl to see if the pH has reached 7.4 yet.
- Wash off tip of probe with DI water in between uses.
Add more double distilled water (ddH2O) until the volume is 1000 mL .
Create the Elution Solution by mixing the following:
- 880 mL of distilled water
- 100 mL of 10 X PBS Solution
- 10 mL of NaPP stock
- 10 mL of Tween 80 stock
- 1 mL of Antifoam A stock.
Autoclave Elution Solution before using.
Appendix II - Prepare Skim Milk Solution
Appendix II - Prepare Skim Milk Solution
Dissolve 5 g of Skim Milk Powder in 100 mL of distilled water using a magnetic stir bar.
Autoclave Skim Milk Solution before using.