National Seed Health System

ARCHIVED 08.03.2017    Cb 1.2 – Acidovorax avenae ssp citrulli – Seminis PCR

PROCEDURE:

PCR SEED WASH PROTOCOL

Caution: Substitution of other primers or materials may significantly alter the results obtained using this protocol. This method is optimized specifically for use with the Seminis WFB1 and WFB2 primers.

DAY 1:

1. Initiate a culture of Xanthomonas campestris pv. vesicatoria on YDC plates and incubate at 28°C. If timing is such that a culture cannot be started two days ahead of the assay date, then one can be initiated one day ahead with heavy streaking on YDC.

2. Initiate 2 tubes of Nutrient Broth with a viable culture of Acidovorax avenae pv. citrulli (Aac) the day before the assay is to be run. Place the tubes on a shaker at 37°C at 200 rpm.

3. Count out 2 samples of 5,000 seeds of the seeds to be used as the negative and positive control and place in Ziplock bags. A number of these bags can be prepared in advance. Label these bags as Sample 19 and 20 and record the lot number and sample numbers on the assay record sheet.

4. The recommended sample size is 30,000 seeds. The maximum subsample size is 5000 seeds. Record the inspection lot and batch numbers on the record sheet. Use a separate disposable container to weigh each lot and clean the weighing area with 10% Clorox between each lot. Any positives obtained in the PCR test will be re-tested at 1X in a greenhouse grow-out test.

5. Place each subsample of 5,000 seeds in a 1.0-gallon freezer Ziplock bag. Place this sealed bag inside another Ziplock bag. Label both bags with the sample number.

6. Calculate the amount of PABST buffer needed for the number of samples to be run the next day. Small-seeded samples (≤250 g) use 300 ml, intermediately sized samples (>250 and ≤400g) use 450 ml and large-seeded samples (>400 g) use 600 ml. Write the number of ml for each lot on the assay record sheet.

7. PABST is “PhosTween” (See below) with the addition of ascorbic acid and potassium metabisulfite. This buffer must be made fresh immediately before use. To expedite the sample prep on day two, weigh out the appropriate amount of ascorbic acid and potassium metabisulfite to be added to the PhosTween on day two. Store the chemicals separately in a plastic cup and cover with foil. Store the chemicals with the seed samples to be run on day two.

PhosTween (No Volume adjustment)
Ascorbic Acid 17.6 g/l PhosTween
Potassium metabisulfite 2.2 g/l PhosTween

DAY 2:
The seed wash technique requires four hours to complete. To expedite DNA extraction, it is best to begin the seed wash process early. Make sure all work surfaces and rotors have been cleaned with alcohol followed by Lysol with 10% Clorox. Wear gloves and use great care to prevent contamination through all of the steps.

1. Prepare PABST buffer by adding the ascorbic acid and potassium metabisulfite to two liters of PhosTween. After the chemicals are dissolved, add the remaining volume of PhosTween needed. Adjust the pH to 6.50 using concentrated NaOH (~5 ml/l of PABST).

2. Prepare a suspension of Xanthomonas campestris pv. vesicatoria (Xcv) in a 50ml disposable sterile centrifuge tube in PhosTween. Use enough material from single colonies on a YDC culture to prepare a faintly turbid suspension. Vortex the suspension to ensure that all of the culture material is in solution. Measure the OD₆₂₀ of this suspension and adjust it to 0.045-0.055.

3. Using the nutrient broth suspension of Aac from the 37°C incubator, vortex and add a small volume to 9ml of PhosTween. Adjust the concentration so that the OD₆₂₀ is between 0.095 and 0.105. Use PhosTween as the reference for the spectrophotometer. Make a 1:10 serial dilution of the OD adjusted Aac suspension to 107 in 9ml tubes of PhosTween.

CAUTION: Wear gloves during this step and discard them after this step is completed. Wear a clean pair of gloves for the next step.

4. Add the required volume of PABST to the seed samples in Ziplock bags, seal both plastic bags and place upright in plastic tubs. After PABST is added to all bags, add 1 ml of the Xcv suspension made in Step 2 to each of the bags in the assay with the exception of #20, the positive control. Make sure all bags are closed tightly.

5. Add 2.3ml of the 10³ Aac dilution tube from Step 3 to sample #20. This is now the Aac positive control. Seal this bag well and place all of the bags in the tubs into the shaker and shake all of the bags for one hour at 150 rpm at room temperature.

6. Spread-plate 0.2 ml of the 10⁶ and 10⁷ dilutions of Aac onto 8 Nutrient Agar plates for each dilution. Also spread-plate 0.2ml of the 10⁶ dilution onto 4 Tween plates. Incubate 24-48 hours at 28°C. After incubation, count colony numbers for calculation of the actual number of cells added to the control seed.

CAUTION: Discard the gloves used for steps 4-6 and wear clean gloves for the following steps.

7. Prepare centrifuge bottles and funnel setup in petri plate boxes. Note that there are specific numbers on the boxes for each position. Centrifuge bottles are set in place. Be certain that the funnels’ ends are set into the centrifuge bottles. Number the caps on the 250ml centrifuge bottles from 1 through 18 with tape and an indelible marker.

8. Wear gloves and always handle the positive control bag (#20) last. Remove the inner bag of seed and place it into the funnels. Make note of any bags that leak. Cut two or three small vertical slits with a razor blade in one bottom corner and let the liquid drain into the bottles. Alternate blades and soak in Lysol/Clorox followed by 95% ethanol between each bag. After the seed is well drained, remove the seed bag and funnel without dripping onto any surfaces. Discard seed in an autoclave bag.

9. Make sure bottles are balanced before centrifuging. Add a small amount of PABST if needed. Centrifuge (SLA-1500 rotor in the Sorvall RC-5B centrifuge) for 5 minutes at 2500 rpm to pellet soil and debris.

10. Transfer the supernatant into a clean 250ml-centrifuge bottle and transfer the numbered cap to the new bottle. Decant slowly so that if the pellet is soft, pieces of the pellet will not accompany the supernatant. If necessary, leave a couple of milliliters of solution in the bottle to avoid particulates. Centrifuge (SLA-1500 rotor in the Sorvall RC-5B centrifuge) the filtrate for 15 minutes at 5,600 rpm to pellet bacteria.

11. Discard the supernatant being careful to preserve the pellet. If necessary, leave a couple of milliliters of solution in the bottle to avoid discarding soft pellet pieces. Add 0.5 g PVPP (prealiquoted in plastic bags) to each centrifuge bottle. Dump the entire contents of a 5ml tube of PhosTween into each centrifuge bottle and resuspend the pellet by gently hand swirling the bottle. Vortex if necessary to resuspend the pellet. Incubate the suspension at room temperature for 30 minutes. Tilt the bottles so that the PVPP is covered completely by the PhosTween.

12. Number 50-ml centrifuge tubes from 1 through 20.

13. For this step, change gloves between each sample! Pour all the liquid from each centrifuge bottle from Step 10 through miracloth filter into the correspondingly numbered 50ml centrifuge tube. Add the entire contents of a 15ml tube of PhosTween buffer to each centrifuge bottle in order to rinse the PVPP. Pour all of the rinsate through miracloth into the correspondingly numbered centrifuge tube. Squeeze remaining liquid from miracloth.

14. Centrifuge the suspension for 15 minutes at 7,000 rpm in the SA-600 rotor. Immediately after the spin is complete, discard the supernatant without disturbing the pellet.

15. Prepare the pellet for DNA extraction in the laminar flow hood. Complete the work with one tube completely before proceeding to the next. This will reduce the chances for cross-contamination. Add 1.3 mls PhosTween (or less if liquid is retained in the tube) to the 50ml centrifuge tube. Resuspend the pellet into the PhosTween by pipetting the buffer into the pellet using a sterile 2ml pipette or by vortexing. Make certain the pellet is completely resuspended and there are no clumps in the solution. Transfer the entire contents to a 2.0ml microfuge tube. This suspension is now ready for DNA extraction. Proceed to the next tube until all tubes are completed. Change pipettes between each sample. Alcohol and Lysol/Clorox hands if a splash occurs.

Clean up
Discard all disposable tips, gloves and seeds in an autoclave bag. Place the autoclave bag in the receptacle for autoclaving. All bottles and liquid must be autoclaved before they go to dishwashing. Spray down all surfaces with alcohol followed by Lysol/Clorox.

DNA EXTRACTION FOR PCR DETECTION

Caution: Substitution of other primers or materials may significantly alter the results obtained using this protocol. This method is optimized specifically for use with the Seminis WFB1 and WFB2 primers.

After the seed wash is complete, take the entire aliquot of the seed wash material and proceed as follows:

Note: Turn ON both water baths (37°C and 65°C) and place two tubes of CTAB into a float and place them into the 65°C water bath. Place one tube of SDS in a float at 37°C. Please wear “Evolution One” or “Safeskin _TM” brand gloves for the first 2 steps, then switch to “Ansell-Edmont” blue nitrile gloves for the rest of the procedure.

DAY 2:

1. Microfuge the sample of seed wash material for 2 minutes at 16,000g to precipitate the cells, remove the supernatant using a 1ml pipette.

2. Re-suspend the pellet in 582 µl of TE. Add 15 µl of 20% SDS and 3 µl of proteinase K and incubate at 37°C for 1-1.5 hours. At this step, it is important to be careful of crossover contamination between samples. After this step is completed, throw your gloves away.

3. Preheat a tube of CTAB/NaCl in the 65°C-water bath. Add 100 µl of 5 M NaCl and mix thoroughly by pipetting up and down a few times. NOTE: Take precautions to avoid crossover contamination of the samples.

4. Add 80 µl of warmed CTAB/NaCl solution and mix thoroughly by pipetting. Incubate the mixture for 15 minutes at 65°C. After this step, remove your gloves and throw them away. Put on a fresh pair of nitrile gloves to start the next step.

5. Add 700 µl of chloroform/isoamyl alcohol (24:1). Pipette up and down a few times to mix the solutions. Place the tubes on a rocker shaker in a fume hood for 15 minutes; then centrifuge for 5 minutes at 16,000g.

6. Remove aqueous supernatant and place in a clean, irradiated 2.0 ml tube.

7. Add 50 µl Phenol/Chloroform/Isoamyl alcohol (25:24:1) with caution. Pipette up and down a few times to mix the solutions. Place the tubes on a rocker shaker in a fume hood for 15 minutes; then centrifuge for 5 minutes at 16,000g.

8. Transfer a maximum of 550 µl of the supernatant to a clean, irradiated 1.5ml centrifuge tube. Avoid the interface of the two phases. Add 550 µl of Isopropanol to precipitate the nucleic acids. Shake the tube.

9. Incubate the sample overnight at -80°C. Clean all laboratory surfaces with Clorox/Lysol.

DAY 3
Prepare the master mix of PCR prior to beginning the DNA preparation to ensure that no contamination occurs. Use Taq-gold (Applied Biosystems), which will not react until the initial heating is completed.

The master mix consists of the following components per reaction:

• 5.0 µl of 10X Applied Biosystems PCR Buffer II
• 8.0 µl DNTPs (1.25mM)
• 3.3 µl 25mM MgCl₂ (Applied Biosystems)
• 1µl primer Seminis WFB 1 (5pmols/µl)
• 1µl primer Seminis WFB 2 (5pmols/µl)
• 0.25µl Applied Biosystems Taq-Gold Polymerase
• 26.45 µl H₂O (5 µl of sample DNA is added for a final volume of 50µl/PCR reaction.)

The primer sequence for WFB 1 and WFB 2 are as follows:

The sequences for RST 65/69 primers (Eur. Jour. Plant Path. 110:285-292) are as follows:

The PCR conditions are as follows:

1. 10 minutes at 95°C
2. 30 seconds at 95°C
3. 30 seconds at 62.1°C
4. 30 seconds at 72°C
5. 35 cycles step 2 to step 4
6. 5 minutes extension at 72°C
7. Hold at 4°C

10. After thawing, spin the DNA samples for 30 minutes at 16,000 g in the microfuge. Remove the supernatant with a drawn-out pipette being careful not to disturb the DNA pellet. Use a separate pipette for each sample to minimize crossover contamination.

11. Wash the pellet with 500 µl of 70% ethanol. Don’t shake the tube. Lightly invert or roll the tube so that the entire inside of the tube gets washed with ethanol. Be careful not to lose the DNA pellet.

12. Centrifuge the samples at 16,000 g for 10 minutes to re-pellet the DNA.

13. Remove the supernatant with a drawn out glass pipette, using a separate pipette for each sample tube. Dry the pellet by letting the tube sit open in the desiccation chamber under a vacuum for approximately 10 minutes. Vent the chamber SLOWLY or risk losing the dry DNA as air rushes into the desiccation chamber.

14. Re-dissolve the pellet in preheated (65°C) TE (100 µl/sample) by incubating the tubes in the 65°C water bath for at least 30 minutes before proceeding.

15. Make a 1:50 dilution of the DNA in water (5µl stock DNA in 245 µl sterile MQ H ₂O). Label and freeze the original DNA at −80°C.

16. Run 50µl of the diluted DNA through a BioRad microspin column to remove inhibitors and further purify the sample. Briefly vortex the suspension.

17. Run two replications for each subsample of DNA. Load 5 µl of each sample into PCR tubes already containing the master mix. Do this in a laminar flow hood. Cap the tubes lightly on the bench-top, then securely once the tubes are in the PCR thermalcycler block. Store the original DNA sample at either -80°C or -20°C (in a non-frost free refrigerator).

18. Initiate PCR conditions on the thermalcycler.

19. While the PCR is running, pour the required size (for the number of samples) 2.0% Seakem LE gel. Use 2.5 g in 125 ml or 7.0 g in 350 ml of 0.5x TBE buffer for the gel. Before the gel is poured, add 2.0 µl (125 ml gel) or 4.0 µl (350 ml gel) of Ethidium Bromide (10 mg/ml) to the hot agarose and swirl the flask to mix.

20. After the PCR cycling is completed, add the proper amount of gel dye solution to each PCR sample. This should be done in an area isolated from the area where seed and DNA extractions are completed. Then load 15 µl of each sample onto the gel.

21. Run the gel for about 1 hour at 80 volts (mini-wide gel) or 1.5 hours at 100 volts (BioRad Model 192 with 350 ml gel).

22. After electrophoresis, view the results on the trans-illuminator and photograph the gel with the Kodak Digital Camera and save the digital image for documentation of the PCR. Record the results of the run on the data record sheet, combine these records with the gel documentation sheet, and place in the BFB PCR result notebook. Enter all final results into SAP.

23. If there is a positive reaction for Aac in the PCR test, then a confirmation test can be performed. Evaluate whether the reaction is as strong as the positive control (spiked sample). If so, then a followup PCR can be run using AAC 1/2 primers (Phytopathology 93:528-534). If the reaction is not as strong as the control sample, then a sample of the seedlot must be submitted for growout test.

24. When running the followup PCR test using AAC 1/2 primers, also run a second PCR using Seminis WFB 1/2 primers. Include all positive and negative control samples as well as the subsamples with positive reactions. Interpretation is dependent upon the Seminis WFB 1/2 primers giving the same response as seen in the original PCR test.

25. If there is a weak response on the Xcv reactions then there may have been inhibition of the PCR reactions. The stored DNA subsamples that were inhibited can be rerun beginning at step 15. Include all controls. Dilute 1:10 after step 16 and PCR both the undiluted and diluted samples using both Seminis WFB 1/2 and Xcv primers.