National Seed Health System

ARCHIVED 08.03.2017    Cb 1.4 – Acidovorax avenae ssp citrulli – Monsanto PCR

PROCEDURE:

I. Preparation prior to assay

A. The following media should be prepared (see below): eight plates of Nutrient Agar and two plates of Tween agar are needed. Prepare and aliquot DNA extraction and PCR reagents.

B. Initiate a culture of Xanthomonas campestris pv. vesicatoria (Xcv) on YDC plates and incubate at 27-29°

C. This culture should be started two to three days ahead of assay date. C. The day before the assay is to be run, inoculate 2-3 tubes of Nutrient Broth with a viable culture of Acidovorax avenae pv. citrulli (Aac). To do this, transfer a colony of Aac from a Tween plate into 9ml of sterile nutrient broth and place tubes in the incubator/shaker at an average temperature of 36-38°C at 150-180 rpm for approximately 16 hours.

D. Seed Sample Prep: The maximum seed sample size per bag is 5000 seeds. Melon and watermelon samples have 5000 seed subsamples and squash seed has 2500 seed subsamples. Samples requiring 30,000 seed testing can be run in the same seedwash.

E. For each sample, count 100 seeds, weigh the 100 seeds and calculate the weight required for the appropriate subsample based on the 100 seed weight. Weigh out the required subsamples (5000 seeds). You can also use the sample thousand seed weight from the sample label. Repeat this step for each sample.

F. Place prepared samples in labeled heat sealable bags. Every test requires a negative and positive control, which should consist of 5000 seeds of a susceptible melon, watermelon, or squash variety.

G. Buffer Preparation: Calculate the amount of PABST buffer required based on the type and weight of the seed. Note: Some samples may require additional buffer to obtain good washing during shaker process. Volumes of buffer per weight are as follows:

The PABST buffer is Phostween with the addition of ascorbic acid and potassium disulfite. Phostween is prepared in advance and stored as a 20X solution. Note: Always prepare a minimum of 200 additional ml of PABST buffer to accommodate the need for additional buffer to bags and to even out centrifuge bottles in later steps.

1. Divide volume needed PABST by 20 to determine the amount of 20X Phostween to add to buffer container. Determine the amount of PABST needed by looking at the final volume on the BFB PCR Prep Sheet and extrapolate for total volumes less than 6.5 liters.

2. Adjust volume with MQ water to dilute Phostween buffer to 1X.

3. Calculate and weigh out ascorbic acid and potassium disulfite.

4. Per liter Phostween, add ascorbic acid 17.6 g and potassium disulfite 2.2 g

5. Dissolve the ascorbic acid in 1X Phostween buffer.

6. IMPORTANT: At this point adjust the pH to 5 with saturated NaOH solution (typically, 36-40 ml), as the addition of potassium disulfite at pH ~4 gives off chlorine gas.

7. In a fume hood, dissolve the potassium disulfite into the 1X Phostween solution.

8. After the chemicals are dissolved, adjust the pH to 6.5}0.05 by adding saturated NaOH solution.

9. Add the required volume of PABST buffer to the seed sample bags with the seed being careful not to let the measuring device come in contact with the seeds.

H. Suspension Preparation Turn on the spectrophotometer to permit time for the machine to warm-up. This will take approximately 5 min. Note: Phostween buffer is stored in 9 ml aliquots. Prior to start, obtain 11 9 ml aliquots, a 50 ml orange centrifuge tube with 40 ml of Phostween buffer, a box of cuvettes for the spectrophotometer, a culture of Xcv streaked onto YDC plates, the tubes of nutrient broth inoculated with Aac in step I.C, and sterile inoculation loops.

1. For Xanthomonas campestris pv. vesicatoria suspension:

a. Fill a 1 ml cuvette with 1X Phostween buffer and place in spectrophotometer. Zero optical density reading at 600 nm (OD600).

b. Make bacterial suspension of Xcv in Phostween making sure to vortex vigorously until the Xcv is fully in solution. Adjust the suspension by either adding bacterial suspension or sterile Phostween such that the final OD is between 0.045 and 0.055 at 600 nm and you have over 1 ml of volume per subsample. Record the OD600.

2. For Acidovorax avenae pv. citrulli suspension:

a. Shake the nutrient broth Aac culture in the light to look for contamination.

b. Pick the tube with no contamination and transfer approximately 800 μl into a 9 ml Phostween tube.

c. Mix in hand, pour to fill line of cuvette, and measure the OD600.

d. Adjust the concentration of Aac in Phostween to an OD600 value between 0.095 and 0.105, and label the tube with the concentration. Record the OD600 value.

e. Make 1:10 serial dilutions of the OD600 adjusted Aac suspension through 10^-7 in the remaining seven tubes of Phostween with the designated Aac 1ml pipette. Label the tubes with the corresponding dilution number (1-7).

f. Save the original Xcv and Aac suspensions, and tubes 3 and 6 from the Aac serial dilution.

1. Take the Xcv suspension and the 10^-3 dilution of Aac suspension to the buffer preparation area. Dispense 1 ml of the Xcv suspension into all The PABST buffer is Phostween with the addition of ascorbic acid and potassium disulfite. Phostween is prepared in advance and stored as a 20X solution. Note: Always prepare a minimum of 200 additional ml of PABST buffer to accommodate the need for additional buffer to bags and to even out centrifuge bottles in later steps. sample bags and the Xcv process control. Dispense 2.3 ml of the Aac suspension into the Aac process control (positive control).

I. Seed Wash: Heat seal the tops of all of the bags and place in the tubs in the shaker and shake all samples for one hour at 150-180 rpm at room temperature.

J. Dilution Plating: While the bags are shaking, spread plate the 10-6 and 10-7 dilutions (tubes 6 and 7) of Aac.

1. Obtain 6 Nutrient Agar plates and 2 Tween plates.

2. Obtain 3 sterile spreading rods.

3. Place 0.2 ml of the 10^-6 dilution onto 3 Nutrient Agar plates and the 2 Tween plates (if required) and spread them with the rods. Label the plates with (-6) and the date. Place 0.2 ml of the 10^-7 dilution onto 3 Nutrient Agar plates and label the plates with (-7) and the date.

4. Let plates rest for ≈ 20 min. Invert and bag, then place in a 27-29°C incubator until colonies are large enough to count. Tween plates will not be counted, rather used to propagate the Aac culture needed in step I.C above.

5. After incubation, count colony numbers, calculate the CFU/ml seed wash and record.

K. Clean the countertop thoroughly with 10% Clorox and 70% ethanol then set up the 250 ml centrifuge bottles and funnels in the boxes for draining seed wash bags. Place the bottles in the corresponding places in the boxes and place the funnels into the bottles. Put the numbered lid on the counter lined up with the funnel and the corresponding 250 ml centrifuge bottle.

L. Once shaking is complete, remove all bags and place them corner first into the corresponding funnel. Obtain two scalpels, one beaker with 10% Clorox, and a beaker with 70% ethanol. Lance the bag to drain the buffer and minimize seed carryover. Alternate blades and soak in the 10% Clorox followed by 70% ethanol between each sample, and carefully blot the scalpel with paper towel after the 70% ethanol and between samples. Squeeze the sample bags to ensure all the buffer has drained out and discard in an autoclave bag.

M. Balance the bottles before centrifuging by adding PABST buffer if needed. Centrifuge for 5 min at 960 rcf to pellet soil and debris.

N. Pour the supernatant into a clean 250 ml centrifuge bottle and transfer the numbered cap to the new bottle. Collect the supernatant slowly to avoid re-suspension of the pellet. At the same time, try to get as much supernatant as possible to keep the sensitivity of the assay. Leave behind any solution that is full of particulates.

O. Rebalance the bottles containing supernatant with PABST if necessary and centrifuge the filtrate for 15 min, 4800 rcf.

P. Discard the supernatant being careful to preserve the pellet. If necessary leave a couple ml of solution to avoid discarding soft pellet pieces. Add 0.45-0.55 g of dry PVPP to each centrifuge bottle and mix until pellet is in solution. Up to 10 ml of Phostween buffer can be added to help the pellet mix with the PVPP. Incubate at room temperature for 30 min.

Q. Obtain 50 ml centrifuge tubes for the number of subsamples being processed and number with the relevant lab number.

R. Transfer the sample from the 250 ml centrifuge bottles into the correspondingly numbered 50 ml centrifuge tube through a miracloth filter. Rinse the 250 ml centrifuge bottle with 15 ml of Phostween buffer and pour through a miracloth filter. Compress the miracloth filter to capture absorbed buffer then change gloves. Repeat for all samples.

S. Balance the 50 ml centrifuge tubes with Phostween and centrifuge the suspension for 15 min at 6950 rcf. Immediately after the spin is complete, discard the supernatant into the waste container without disturbing the pellet.

T. Prepare a rack of 2.0 ml microcentrifuge tubes for the samples. To the pellet in the 50 ml centrifuge tube, add enough Phostween buffer to completely resuspend the pellet without exceeding a 2.0 ml volume. Transfer the resuspended contents to the corresponding numbered 2.0 ml tube. Do this for all samples and proceed on to the DNA extraction steps(section V below).

U. Completely clean the sample bag preparation and seed wash areas with 70% alcohol followed by 10% Clorox solution. Put used culture plates, collected supernatants, bacterial suspensions and other associated waste in doubled up red autoclave bags. Autoclave the bags and autoclave all reusable labware.

II. Improved DNA Extraction Method (To be used with MO BIO Monsanto Custom Kit Part # 21000-100-MON)

A. Preparation: Turn ON one heat block to 65oC and one water bath to 55°C. Warm PF1 and PF3 for 10 minutes in the water bath at 55°C prior to use. PF1 and PF3 can be used while still warm. Centrifuge the 2.0 ml tubes from step I.T above for 2 min at 16,000g.

B. DNA extraction:

1. Remove the supernatant using a 1ml pipette. Collect supernatant to be autoclaved.

2. Resuspend the seed pellet in 450 μl of warm solution PF1.

3. Transfer resuspended pellet to the corresponding MicroBead Tube containing the MicroBeads (as provided in the kit). Incubate at 65°C for 10 minutes.

4. Secure the MicroBead Tube horizontally to a MO BIO Vortex Adapter.

5. Vortex at maximum speed for 10 minutes.

6. Centrifuge the tubes at 13,000 x g for 1 minute at room temperature. CAUTION: Be sure not to exceed 13,000 x g or tubes may break.

7. Transfer the supernatant to a clean 2 ml collection tube (provided in kit).

8. Add 100 μl of solution PF2 and vortex briefly to mix. Incubate at 4°C for 5 minutes.

9. Centrifuge the tubes at 13,000 x g for 1 minute at room temperature.

10. Avoiding the pellet, transfer the entire volume of supernatant to a clean 2 ml collection tube (provided).

11. Add 900 μl of solution PF3 and vortex to mix. Check solution PF3 for precipitation prior to use. Warm if necessary. Solution PF3 can be used while still warm. NOTE: If using the MO BIO PowerVac Manifold Mini system to wash the spin filter instead of centrifugation, proceed to step II.C instead of step 12.

12. Load 650μl of supernatant onto a spin filter and centrifuge at 13,000 x g for 1 minute. Discard the flow through and repeat until all the supernatant has been loaded onto the spin filter. A total of two to three loads for each sample processed are required.

13. Place the spin filter basket into a clean 2 ml collection tube (provided).

14. Shake to mix solution PF4 before use. Add 650μl of solution PF4 and centrifuge at 13,000 x g for 1 minute at room temperature.

15. Discard the flow through and add 650μl of solution PF5 and centrifuge at 13,000 x g for 1 minute at room temperature.

16. Discard the flow through and centrifuge again at 13,000 x g for 2 minutes to remove residual wash.

17. Place the spin filter basket into a clean 2 ml collection tube (provided).

18. Add 100 μl of solution PF6 to the center of the white filter membrane.

19. Centrifuge at 13,000 x g for 1 minute.

20. Discard the spin filter basket. The DNA is now ready for any downstream application. Proceed to step II.C (Vacuum Protocol) using the PowerVac™ Manifold.

C. Vacuum protocol:

1. For each sample lysate, use one spin filter column. Keep the spin filter in the attached 2 ml collection tube and continue using the collection tube as a spin filter holder until needed for the Vacuum Manifold Protocol. Label each spin filter column to maintain sample identity. If the spin filter becomes clogged during the vacuum procedure, switch to the procedure for purification of the DNA by centrifugation. Follow instructions in step II.C.8 before removing the column from the manifold. You will need to provide 100% ethanol for step II.C.5 of this protocol.

2. For each sample, attach one aluminum PowerVac™ Mini Spin Filter Adapter (MO BIO Catalog# 11992-10 or 11992-20) into the Luer-LokR fitting of one port in the manifold. Gently press a spin filter column into the PowerVac™ Mini Spin Filter Adapter until snugly in place. Ensure that all unused ports of the vacuum manifold are closed. Note: Aluminum PowerVac™ Mini Spin Filter Adapters are reusable.

3. Turn on the vacuum source and open the stopcock for the ports with a filter column installed. For each sample, transfer 650 μl of prepared sample lysate (from step II.B.11) to the respective spin filter column.

4. Continue until all of the lysate has been loaded onto the spin filter column.

5. Turn off the vacuum source and open an unused port to vent the manifold. If all 20 ports are in use, break the vacuum at the source. Load 800 μl of 100% ethanol into the spin filter so that it completely fills the column. Close the unused port and turn on the vacuum. Allow the ethanol to pass through the column completely.

6. Shake to mix solution PF4. Add 650 μl of solution PF4 to each spin filter and wait until solution PF4 has passed through the spin filter completely. Continue to pull a vacuum for another minute to dry the membrane.

7. Add 650μl of solution PF5 to each spin filter and wait until solution PF5 has passed through the spin filter completely. Continue to pull a vacuum for another minute to dry the membrane.

8. Turn off the vacuum source and open an unused port to vent the manifold. If all 20 ports are in use, break the vacuum at the source. Make certain that all vacuum pressure is released before performing the next step. It is important to turn off the vacuum at the source to prevent backflow into the columns.

9. Remove the spin filter column and place in the original labeled 2 ml collection tube. Place into the centrifuge and spin at 13,000 × g for 2 minutes to completely dry the membrane.

10. Transfer the spin filter column to a new 2 ml collection tube and add 100μl of solution PF6 (TE) to the center of the white filter membrane.

11. Centrifuge at room temperature for 1 minute at 13,000 x g.

12. Discard the spin filter column. The DNA in the tube is now ready for any downstream application. Proceed to step II.D, Real-time PCR Loading and Analysis.

D. Real-time PCR Loading and Analysis:

1. For PCR, make a 1:50 dilution of the DNA in water by mixing 5 μl stock DNA and 245 μl sterile MQ water in a labeled 1.5 ml tube. Store the original DNA solution at -20°C or long term at -80°C.

2. Primer/probes are stored in aliquots in the -20oC freezer. Obtain sterile H2O, primer/probe aliquots, and the ABI 2x Universal Master Mix. Following the master mix recipe for qPCR below, make enough reactions for your samples plus 2 extra.

The primer/probe sequences for detecting Aac are as follows:

The primer/probe sequences for detecting Xcv are as follows:

The Master Mix Recipe for qPCR (per one reaction):

Component 1X  25ul

H₂O  7ul

2X ABI Universal MM  12.5ul

Forward Primer (100uM)  0.225ul

Reverse Primer (100uM)  0.225ul

Probe (100uM)  0.062ul

Template DNA  5ul

3. After the master mix is made, dispense it into the designated PCR plate as specified by the appropriate plate template. This is done by using the mastermix only repeater pipette.

4. Run 2 replications for each subsample of DNA. Load 5 μl of each sample into the corresponding well on the PCR plate already containing the master mix. Seal the top of the PCR plate with the press sealer and spin the plate down in the plate spinner. Initiate the real-time PCR program on the Roche LC-480 or ABI Viia7 units under the following protocol:

Step 1: 10 min, 95°C

Step 2: 15 sec, 95°C

Step 3: 1 min, 60° C

Step 4: 40 cycles of steps 2-3

Step 5: 10 second extension, 40°C

For the Roche LC-480 unit, analyze qPCR data by fit points method and set the curve threshold above the negative background. Calculate the Aac section separate from the Xcv internal control, and export all data into the appropriate folders and spreadsheets according to training.

E. Interpretation of Results:

1. Real-time PCR: A real-time PCR run is considered valid if there is no fluorescence produced by the negative controls, and all positive controls result in expected Ct values. For real-time PCR, the Xcv positive control should produce a Ct value below 28 and the Aac positive control should have a Ct of approximately 30.

2. Samples that did not produce Xcv amplicons: If there was no amplification observed in these samples, this can be indicative of inhibition due to seed type or quality. Any sample not producing an Xcv band, or on the real-time PCR with a Ct above 28 will need to be tested via grow out for assessment. a. Samples that did produce Xcv amplicons: The Aac PCR results can be evaluated for the presence of Aac. Any positives obtained in the PCR or real-time PCR test will be tested at 1X in a greenhouse grow-out test.

III. DEFINITIONS

MQ: water refers to water purified with the Milli-Q system.

RO: water refers to water purified by reverse-osmosis.

RECIPES:

Modified Tween Medium for Bacterial Fruit Blotch

Stir well to dissolve the Tween 80. Adjust pH to 7.3 using NaOH. Autoclave for 25 min, cool then add the following antibiotics.

Nutrient Agar:

Nutrient Broth:

General Bacterial Medium (YDC):

*If pouring by hand add 1 drop of Pourite per liter of materials

PhosTween buffer-20X Stock Solution

In 800ml MQ H₂0 dissolve:

Per 1 Liter

Potassium Phosphate (K₂HPO4•3H₂O)   39.2g

Potassium Phosphate (KH₂PO₄•)   19.88 g

Tween 20      4 ml

Adjust total volume to 1.0 liter. Adjust the pH using 5 M HCI to a final pH of 6.5.

Dilute buffer 1/20 in MQ or RO water to make lX concentration.

5.0M NaCl: Sodium Chloride

Dissolve 29.22 g of NaCl (VWR cat.# VW 6430-7) in 80 ml of MQ water. Adjust the volume to a total of 100 ml.

CTAB/NaCI: Hexadecyltrimethylammonium Bromide/Sodium Chloride solution.

Dissolve 4.lg NaCl (VWR cat.# VW 6430-7) and 10 g CTAB (Sigma cat.# H- 6269) in 100 ml of MQ water. Mix reagents on a stir plate with mild heat until the crystals are completely dissolved.

Caution: CTAB is a highly toxic compound, be sure to weigh out and add this chemical to water while in the flow hood.

TE buffer: Tris-EDTA

Dissolve 0.394 g Trizma Hydochloride (Sigma cat. # T-7419) and 1.0 ml of 0.25M EDTA stock in 250 ml MQ water. ADjust the pH to 8.0 using 1.0 M NaOH.

70 % Ethanol:

Add 70.0 ml of 100% ethanol to 30.0 ml of MQ water in the hood and invert to mix.

20% SDS: Sodium Dodecyl Sulfate

(This reagent is found as Lauryl Sulfate disodium salt (Sigma cat. # L-4390).

Dissolve 5.0 g SDS in 25.0 ml of MQ water. In order to mix this reagent properly you must add a stir bar to the bottle and stir on a hot plate over medium-low heat.

Caution: SDS is a toxic compound, be sure to weigh out and add this chemical to water while in the flow hood.

Phenol·Chloroform·lsoamyl Alcohol:

This reagent comes from Sigma (cat.# P-2069) with its own equilibration buffer.

Add the equilibration buffer to the bottle of Phenol-Chloroform­Isoamyl Alcohol, invert several times to mix, and let the bottle equilibrate in the negative pressure hood for approx. 2 h.

6X Gel loading dye:

This reagent comes prepared from Sigma ( cat. # G-7 654 ).

Proteinase K:

This reagent comes prepared from Sigma (cat.# P-2308). The reagent comes as a lyophillized powder in 100 mg quantity.

Add 5.0 ml of sterile MQ water to the vial, cap tightly, and invert several times to bring the powder to solution. This is a 20 mg/ml stock ready for aliquotting or direct use.

lsopropanol:

This reagent comes prepared from Sigma (cat.# l-9516). The reagent comes as a lyophillized powder in 100 mg quantity.

PVPP: Polyvinylpolypyrrolidone (Sigma cat. # P-6755). This protocol is taken from Holben et al., 1988. (Appl. Environ. Microbiol. 54:703-711). Wear gloves on all steps and follow safety precautions with all materials.

1. Mix 450.0 g of PVPP in 4 l of 3.0M HCl. The HCl is prepared by diluting concentrated HCl which has an initial molarity of 12.1. (3.0M/11.6M x 4.0 l = 0.992 l of HCl added to 3.008 of MQ water.)

2. Mix and let sit at room temperature in a fume hood for 12-16 h with occasional gentle stirring.

3. Filter sterilized the suspension through Miracloth (Calbiochem #475855). Gentle squeezing of the Miracloth can be done to remove most of the liquid. Eventually, the Miracloth will tear, be careful not to lose any PVPP.

4. Resuspend the PVPP in 4.0 l of 20mM potassium phosphate buffer, pH=7.4. Prepare the buffer by adding 10.89 g of KH₂PO4 to a total of 4.0 l of MQ water. Adjust the pH to 7.4 using 8.0M KOH (~10 ml).

5. Stir at room temperature for 1-2 min.

6. Pass the suspension through Miracloth as in step 3.

7. Repeat steps 4 through 6 until the pH of the suspension reaches 7.4 (8-10 times)

8. Spread the PVPP out into a thin layer on aluminum foil and dry in a flow hood overnight. Material can be stored in a capped jar and is useful for extended periods of time, >1 year. Starting with 450 g, yield should be ~400 g after the procedure is complete.