Skim Milk Flocculation and RNA Extraction for SARS-CoV-2 Viral Capture

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 0h 0m 30s .

Place Moore swab sample in a clean stomacher bag with 100mL 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.

Repeat Steps 2.1 - 2.4 until you have 300mL of sample volume.

If using the potato ricer method:

Place Moore swab sample in a potato ricer and squeeze all the liquid out into a 600 mL beaker.

Pour the squeezed liquid into a labeled 500 mL Erlenmeyer flask.

Place the Moore swab back in the beaker. Add 100mL 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.

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 300mL 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.

Wipe down the benchtop scale after use using 10% bleach followed by 70% ethanol.

Centrifuge for 0h 15m 0s at 5000rpm,0h 0m 0s.

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.

Pour only the supernatant back into the flask (should be approximately 250mL).

Adjust the pH of the sample to 3.5 using 5% HCl and 5% NaOH using a pH probe.

Aliquot 10µL 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.

Add 1% volume of Skim Milk Solution (see Appendix II for preparation steps) to the sample volume.

Place aluminum foil over the opening of the flask.

Place sample on shaker 120rpm .

Pour sample from flask into sterilized and labeled centrifuge bottle.

Centrifuge sample for 0h 30m 0s at 12000rpm,0h 0m 0s.

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 800µLof from the Qiagen RNeasy Mini Kit (Cat. No. 74106) into the centrifuge bottle.

Mix well by pipetting the pellet and up and down about 30 times or until the mixture looks homogeneous.

Pipette 800µL of homogenized mixture into a labeled 2 mL DNA LoBind microcentrifuge tube.

Centrifuge tube for 0h 3m 0s at full speed.

Transfer all of the sample into a new labeled tube.

Aliquot 800µL of 70% molecular ethanol.

Transfer 700µL of the sample mixture into a labeled RNeasy spin column. Centrifuge for 0h 0m 30s at full speed. Discard filtrate. Repeat until all of the sample is filtered through the spin column.

Aliquot 700µL of to the RNeasy spin column. Centrifuge for 0h 0m 30s at full speed. Discard filtrate.

Aliquot 500µL of to the RNeasy spin column. Centrifuge for 0h 0m 30s at full speed. Discard filtrate.

Add 500µL of to the RNeasy spin column. Centrifuge for 0h 2m 0s at full speed. Discard filtrate.

Transfer spin column to new 2 mL collection tube and centrifuge for 0h 1m 0s at full speed. Discard filtrate and collection tube.

Place the RNeasy spin column into a 1.7 mL labeled microcentrifuge tube. Aliquot 50µL of (from RNeasy kit). Incubate for approximately 0h 1m 0s at room temperature.

Centrifuge for 0h 1m 0s at full speed.

Aliquot another 50µL of into the the RNeasy spin column in the 1.7 mL labeled microcentrifuge tube. Incubate for approximately 0h 1m 0s at room temperature.

Centrifuge for 0h 1m 0s at full speed.

Close the cap of the 1.7 mL tube containing the final RNA (you will have a final volume of 100µL).

Store RNA at -20°C until it is ready for PCR.

Prepare the Tween 80 stock by dissolving 1mL of in 100mL of distilled water.

Prepare the Antifoam A stock by dissolving 1mL of Antifoam A in 100mL of distilled water.

Prepare the NaPP stock by dissolving 1g of NaPP in 100mL of distilled water.

Prepare the 10X PBS Solution

Mix together:

• 80gNaCl
• 2gKCl
• 14.4g Na₂HPO₄
• 2.4g KH₂PO₄
• 800mL Ddwater

Adjust the solution to 7.4 by adding 5% NaOH and/or 5% HCl.

Add more until the volume is 1000mL .

Create the Elution Solution by mixing the following:

• 880mL of distilled water
• 100mL of 10 X PBS Solution
• 10mL of NaPP stock
• 10mL of Tween 80 stock
• 1mL of Antifoam A stock.

Autoclave Elution Solution before using.

Dissolve 5g of in 100mL of distilled water using a magnetic stir bar.

Autoclave Skim Milk Solution before using.