WinterVomitingLab:Protocols/Virus Removal from Berries Using Sanitizers

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Overview

• Removal of virus from berries using a liquid sanitizer solution.

Materials

• Berries
• Sanitizer(s)
• Elution buffer
• Neutralization buffer
• Centrifuge bottles
• Sterilized paper towels

Procedure

1. Weigh 20-30g samples of berries and UV both sides in a hood for 10-15 minutes.
2. Prepare sanitizer solutions.
3. For samples-
   1. Surface inoculate berries with 100 μl of prepared virus stock and allow to dry (~40-60 minutes).
   2. When dry, place berries in a beaker containing 110 ml of sanitizer solution for the appropriate exposure time.
   3. After treatment, decant solution into waste container and transfer berries to a container with 100 ml PBS. Rinse and immediately decant solution into waste container.
   4. Transfer berries to pre-sterilized paper towel(s) and gently pat dry.
   5. Transfer berries to a centrifuge bottle containing 110 ml elution buffer (high salt PBS w/ 10% tween 20) and swirl container gently by hand for one minute.
   6. Centrifuge at low speed (2,500-3,000 x g) for 10 minutes at 4°C.
   7. Separate debris from the liquid sample using a 100 μm cell strainer and make the following aliquots- one 30 ml, two 40 ml, and two 500 μl. *30 and 40 ml aliquots are for use in PEG precipitation if needed.
   8. Store samples at -70°C until further processing.
4. For neutralization controls-
   1. Place uninoculated berries into a beaker containing 110 ml of sanitizer solution for the appropriate exposure time.
   2. After treatment, decant solution into waste container and transfer berries to a container with 100 ml PBS. Rinse and immediately decant solution into waste container.
   3. Transfer berries to pre-sterilized paper towel(s) and gently pat dry.
   4. Transfer berries to a centrifuge bottle containing 110 ml elution buffer (high salt PBS w/ 10% tween 20) and swirl gently by hand for one minute.
   5. Remove 1000 μl of elution buffer and set aside to use as a negative control.
   6. Remove berries from elution buffer and inoculate now neutralized sanitizer with 100 μl prepared virus stock.
   7. Centrifuge at low speed (2,500-3,000 x g) for 10 minutes at 4°C.
   8. Separate debris from the liquid sample using a 100 μm cell strainer and make the following aliquots- one 30 ml, two 40 ml, and two 500 μl. *30 ml and 40 ml aliquots are for use in PEG precipitation if needed.
   9. Store samples at -70°C until further processing.
5. For negative controls-
   1. See step 4-5 above.
6. For recovery control without rinse step-
   1. Surface inoculate berries with 100 μl of prepared virus stock and allow to dry (~40-60 minutes).
   2. Transfer berries to a centrifuge bottle containing 110 ml elution buffer (high salt PBS w/ 10% tween 20) and swirl gently by hand for one minute.
   3. Centrifuge at low speed (2,500-3,000 x g) for 10 minutes at 4°C.
7. For recovery control with rinse step-
   1. Surface inoculate berries with 100 μl of prepared virus stock and allow to dry (~40-60 minutes).
   2. Transfer berries to a container with 100 ml PBS. Rinse and immediately decant solution into waste container.
   3. Transfer berries to pre-sterilized paper towel(s) and gently pat dry.
   4. Transfer berries to a centrifuge bottle containing 110 ml elution buffer (high salt PBS w/ 10% tween 20) and swirl gently by hand for one minute.
   5. Centrifuge at low speed (2,500-3,000 x g) for 10 minutes at 4°C.
   6. Separate debris from the liquid sample using a 100 μm cell strainer and make the following aliquots- one 30 ml, two 40 ml, and two 500 μl. *30 ml and 40 ml aliquots are for use in PEG precipitation if needed.
   7. Store samples at -70°C until further processing.
8. For positive controls-
   1. For each experiment, freeze 100-200 μl of the virus stock used to inoculate the berries. This will serve as the positive control and as a means of calculating the inoculum concentration.
   2. Perform a serial dilution of the virus stock just before plaque assay.
9. Plaque assay controls (performed with each plaque assay to aid in troubleshooting if questionable results are obtained)-
   1. Negative control with agar
   2. Negative control without agar (liquid culture)
   3. Positive control (-2 dilution of virus stock) with agar
   4. Positive control (-2 dilution of virus stock ) without agar (liquid culture)
10. After the plaque assay is performed with one of the 500 μl aliquots of sample (if negative results are obtained with the undiluted samples), the remaining frozen aliquots (30 ml, 40 ml, and 40 ml) will need to be concentrated and assayed. Refer to the PEG precipitation protocol if necessary.

References

Relevant papers and books

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