DAS-ELISA kit

For the detection ofRalstonia solanacearum

in soil

INTERNATIONAL POTATO CENTER (CIP)2001

INSTRUCTIONS FOR USE

DAS-ELISA kitFor the detection ofRalstonia solanacearumin soil

INSTRUCTIONS FOR USE

Dr Sylvie PriouInternational Potato Center (CIP)2001

© 2001 Centro Internacional de la Papa, Lima, Peru. All rights reserved

Contents

Page

Introduction 5

Materials 6

Sample preparation 8

Serological test 11

Sources/suppliers of the products included in the kit 15

List of supplies included in the kit 16

AppendicesAppendix 1. Preparation of the enrichment broth (1x) 18Appendix 2. Protocol for sample collection 18Appendix 3. Sample size 19

Coat the wells of a microtitration platewith ( )-specific

rabbit immunoglobulins (IgG)Ralstonia solanacearum Rs

Microtitration plate

Rs-specific bbitimmunoglobulins (IgG)

Rs in soil solution

Enzyme-labeled -specific rabbit IgG(conjugated IgG)

Rs

Enzyme substrate

Add soil solution; in the soil will bind to the IgGRs

Add enzyme-labeled -specific rabbit IgG;these will bind to the -IgG complex to

form the double antibody sandwich

RsRs

Add the enzyme substrate;if is present a color will developRs

ProcedureFigure 1: The four step ps of the DAS-ELISA

INTRODUCTION

DAS-ELISA (double antibody sandwich enzyme-linked inmunosorbent assay) is an immuno-enzymatic assay that uses a microtitration plate as a support for the samples and reagents.

The procedure for the detecting Ralstonia solanacearum in soil, summarized in Figure 1,consists of:

1. Coating a microtitration plate with Ralstonia solanacearum (Rs)-specific rabbitimmunoglobulins (IgG)

2. Adding solutions prepared from soil samples to the wells of the microtitration plate3. Adding enzyme-labeled Rs-specific rabbit immunoglobulins (conjugated IgG)4. Adding the enzyme substrate; if Rs is present in the soil solution this produces a color

reaction, the intensity of which is proportional to the bacterial concentration

The bacteria may be present in soil at only low concentrations, so before performing theDAS-ELISA an enrichment procedure must be carried out to allow the bacteria to multiply todetectable levels. This is done by incubating the soil solution in an enrichment broth at 30°Cfor 48 hours with constant agitation. By this procedure the method is capable of detectingoriginal bacterial concentrations as low as 20 bacteria per gram of soil (instead of 107

bacteria/g without enrichment).

All races, biovars and serotypes of Rs can be detected with the Rs-specific immunoglobulinsprovided in the kit. Some saprophytic bacteria may cross-react with the antibodies producedagainst Rs when in pure culture at concentrations equal to or higher than 108 bacteria/ml.However, after enrichment of soil solutions containing these saprophytic bacteria, no cross-reactions have been detected in DAS-ELISA.

This kit can be used to monitor Rs in bare soil after harvest as well as in the rhizosphere,rhizoplane and roots of potato or other hosts, to study the epidemiology of the disease, toevaluate the effectiveness of a control method in reducing soil populations of the pathogen,and to determine the suitability of a field for seed production.

Before using the DAS-ELISA kit, read the following instructions carefully and be sure thatyou have all materials ready.

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6

MATERIALS

As soon as you receive the kit, wrap it in a plastic bag and store it in arefrigerator at 4°C. The products are stable for six months. The supplies included in thekit are sufficient for testing six microtitration plates of approximately 40 samples each (forsample size see Appendix 3 on Page 19).

The supplies included in the kit are listed on page 16. The following equipment and materialsare also needed for the assay but are not included in the kit (everything must be veryclean):

To collect soil samples and prepare soil solutions

• Small hand hoe• Sodium hypochloride• Polyethylene bags for collecting soil samples• A bunsen burner or alcohol to disinfect lab tools• A sterile 250-ml erlenmeyer for each soil solution• A hammer for disintegrating soil clumps• A marker or wax pencil to mark the sample bags and plates• A spatula• One 1000-ml graduated cylinder• Two 1000-ml and one 2000-ml flasks (bottles or erlenmeyers) to prepare and storebuffers• Fifty liters of distilled water (alternative: clean boiled rain water)• A pH meter• Test tubes• Vortex

For the enrichment procedure (all must be sterile)

• One sterile 1000-ml flask or erlenmeyer• One sterile 1000-ml graduated cylinder

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• Three liters of sterile distilled water to dilute the enrichment broth• An incubator shaker at 30°C• Five sterile 25- or 50-ml erlenmeyers for each sample• One sterile 10-ml pipette

To perform the ELISA

• One 25- or 50-ml beaker• One 100-ml graduated cylinder• Two-and-half liters of sterile distilled water• An adjustable 100–1000-µl micropipetor or 50–300-µl multipipetor and sterile

corresponding tips, or Pasteur pipettes• A refrigerator• An incubator at 37°C• One 1000-ml erlenmeyer or bottle• One 500-ml erlenmeyer or bottle• Three 125-ml containers or bottles• One 250-ml wash bottle

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SAMPLE PREPARATION

The procedure for soil extraction and sample enrichment is summarized in Figure 2.

Prepare and store buffers in clean flasks. Autoclave (20 minutes at 120°C) the buffers afterpreparation. Store in a refrigerator.

The first time you prepare a buffer solution measure its pH with a pH meter, and then addHCl (bottle #15) or NaOH (bottle #16) as necessary to achieve the desired pH. Write thevolumes of HCl or NaOH added in your manual for faster future applications (if the samewater quality is used, there is no need to check the pH each time).

Preparation of the extraction buffer pH 7.4 (2000 ml)

Dissolve the contents of one packet #4 in 2000 ml distilled water while agitating. Adjust pHto 7.4 if necessary. Autoclave (20 minutes at 120°C) and store.

Preparation of the citrate buffer pH 5.6 (1000 ml)

Dissolve the contents of one packet #14A and one packet #14B in 1000 ml distilled waterwhile agitating. Adjust pH to 5.6 if necessary. Autoclave (20 minutes at 120°C) and store.

Preparation of the soil solution

Sample collection is described in Appendix 2, page 18, and sample size in Appendix 3,page 19.

• Mix 10 g of soil with 90 ml of extraction buffer• Agitate at 180 rpm for 30 minutes at room temperature• Allow the soil suspension to settle for 40 seconds• For a semi-quantification of populations of Rs in soil, make serial 10-fold dilutions up to

10–5 by mixing 1 ml of the supernatant (soil solution) with 9 ml of citrate buffer• For qualitative evaluation of soil inoculum (presence or absence of Rs), make dilutions

to 10–1 and 10–2 by mixing 1 ml of the supernatant (soil solution) with 9 ml of citratebuffer

Figure 2: Soil extraction and enrichment process for detection of in soil by DAS-ELISARalstonia solanacearum

SERIAL 10-FOLD DILUTIONS IN CITRATE BUFFER

SOIL EXTRACTION

DAS-ELISA

ENRICHMENT

Incubation at 30º C for 48 hours under constant agitation

Agitation 30 miSettling 40 seconds

90 ml PBS bu+

+

ffer

10 g soil

Dilution 1/10

9 ml of CIP enrichment broth

Dilution 1/10

Dilution 1/10.

.

.

.

.

.

10-1 10-2 10-5

1 ml of diluted soil extract

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Enrichment procedure (48 hours)

• Dilute 50 ml of the 10x enrichment broth (one bottle #11) with 450 ml of sterile distilledwater

• Dispense 9 ml of the diluted (1x) enrichment broth in each of five sterile 25- or 50-mlerlenmeyers

• Add 1 ml of a different dilution of the soil solution to each erlenmeyer of enrichmentbroth

• Incubate 48 hours at 30°C with constant agitation (180 rpm) in an incubator shaker• At the end of the incubation time, continue with the ELISA test, or store 1 ml of enriched

soil solution in an eppendorf tube at –20°C for later use

The composition of the enrichment broth (1x) is given in Appendix 1, page 18.

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SEROLOGICAL TEST

All volumes and quantities indicated (except for the phosphate buffersaline (PBS)) are for a test using one microtitration plate. For each plateyou will need exactly 250 ml of PBS buffer.

1. Coating the microtitration plate with Rs-specific rabbitimmunoglobulins (IgG)

1.1. Preparation of the coating buffer pH 9.6 (100 ml)

Dissolve the contents of packet #1 in 100 ml of distilled water.

1.2. Preparation of the coating solution (12.5 ml)

To 12.5 ml of the coating buffer add 100 µl (0.1 ml) of Rs-specific rabbit IgG (from eppendorftube #7). Mix well, but gently, avoiding the formation of a foam.

1.3. Incubation with the coating solution

Add 125 µl (0.125 ml) of the coating solution to each well of the microtitration plate; ensurethat all wells are filled. Cover the plate with a piece of parafilm to prevent evaporation andincubate at 37°C for 4 hours. This process will allow immunoglobulins to adhere to thesurface of the wells in the plate.

2. Application of the samples to the wells of the microtitration plate

2.1. Buffer preparation: phosphate buffer saline (PBS) pH 7.4 (1000 ml)

Dissolve the contents of one packet #2 in 1000 ml of distilled water. Mix thoroughly andadjust pH to 7.4.

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2.2. Preparation of the washing buffer (500 ml)

Mix 250 µl (0.25 ml) of Tween-20 (eppendorf tube #3) with 500 ml of PBS buffer. (Tween-20 is very viscous and should be taken up slowly into the micropipetor.)

2.3. Washings

Discard excess coating solution from the microtitration plate, and wash the plate with washingbuffer as follows:

• Fill wells with washing buffer• Leave for 3–4 minutes• Empty the plate• Repeat this process three times• After the last washing put the plate upside down on a paper towel and tap it several

times to remove all buffer

2.4. Incubation of the samples

Plan the placement of the samples on the microtitration plate such that soil samples andcontrols can be easily identified.

With a micropipetor, transfer 125 µl (0.125 ml) samples of one of the enriched soil solutionsto each of two wells of the microtitration plate. Repeat this process for all the other enrichedsoil solutions.

Add the positive and negative controls to each plate. The positive controls (eppendorf tube#12) are boiled suspensions of Rsiat 108, 107, 106 and 105 bacteria/ml and the negativecontrol (eppendorf tube #13) is an enriched extract of Rs-free soil.

Seal the plate with parafilm and incubate it at 4°C in a standard refrigerator for 18 hours(overnight). Rs present in the samples will bind with the immunoglobulins.

3. Adding enzyme-labeled Rs-specific rabbit immunoglobulins(conjugated IgG)

3.1. Washings

Wash the plate as previously described (section 2.3). Repeat washing until plate is completelycolorless.

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3.2. Preparation of the conjugate buffer (12.5 ml)

During the last washing, dissolve the contents of one packet #5 (conjugate buffer) in 12.5ml of washing buffer.

3.3 Preparation of the conjugate solution

While agitating, add 100 µl (0.1 ml) of Rs-specific rabbit IgG conjugated to alkalinephosphatase (conjugated IgG) from eppendorf tube #8 to the 12.5 ml of conjugate buffer.

3.4. Incubation with the conjugate solution

After discarding the last washing buffer, add 125 µl (0.125 ml) of the conjugate solution toeach well of the plate. Seal the plate as before and incubate it at 37°C for 4 hours.

4. Color development (enzymatic reaction)

CAUTION: The substrate tablets and the substrate buffer are highly toxic.Wear gloves when preparing these solutions.

4.1. Preparation of the substrate buffer pH 9.8 (12.5 ml)

Mix 2.5 ml of substrate buffer (bottle #6) with 10 ml of distilled water. Mix thoroughly.Adjust pH to 9.8 if necessary.

4.2. Washings

Discard the conjugate solution from the plate and remove the unbound conjugated IgG bywashing the plate with washing buffer as previously described (section 2.3).

4.3. Preparation of the color development solution

During the last washing step, dissolve one tablet (5 mg) of substrate p-np (p-nitrophenylphosphate) from packet #9 in the 12.5 ml of substrate buffer.

4.4. Incubation with the color development solution

After discarding the last washing buffer, add 125 µl (0.125 ml) of the color developmentsolution to each well of the plate. Add the solution to all the wells as quickly as possible.Leave the plate for 60 minutes at room temperature (20–25°C) for the color to develop.

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4.5. Interpretation of the results

In positive samples, the enzymatic reaction gives rise to a yellow coloration, ranging inintensity between light and dark depending on the concentration of Rs in the samples(observe the plate with white paper underneath). Therefore the most concentrated control(Rs in citrate buffer suspension at 108 bacteria/ml) should be dark yellow, and the leastconcentrated control (105 bacteria/ml) should be light yellow. The color can be measuredwith a spectrophotometer at 405 nm wavelength. The samples are considered positive if theabsorbance is three times as much as the average of the enriched soil solution free of Rsused as the negative control.

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SOURCES/SUPPLIERS OF THE PRODUCTS INCLUDED IN THE KIT

Product Supplying company Minimum quantity(and catalog number) supplied by company

Bacitracin Sigma (B-0125) 0.75 g

Bacto peptone Difco (0118-17-0) 500 g

Benlate 50% Farmagro 1 kg

Casamino acids Difco (0230-01-1) 500 g

Chloramphenicol Sigma (C-0378) 5 g

Citric acid Sigma (C-1909) 500 g

Conjugate (Rs-IgG) CIP

Crystal violet Sigma (C-3886) 25 g

Cycloheximide Sigma (C-7698) 5 g

Dextrose Fisher (D16-500) 500 g

Diethanolamine Fisher (D-45-500) 500 ml

Hydrochloric acid – fuming, 37% (HCl) Merck (317) 2.5 liters

Milk powder (non-fat) Market

p-nitrophenyl phosphate Sigma (N-9389) 50 tablets (5 mg each)

Penicillin-G Sigma (P-7794) 6.35 g

Polymyxin B sulphate Sigma (P-1004) 1 g

Polyvinyl pyrrolidone (PVP-40,000) Sigma (PVP-40) 500 g

Potassium chloride (KCl) Fisher (BP366-500) 500 g

Potassium hydrogen phosphate (KH2PO4) Fisher (BP363-500) 500 g

Rs-IgG CIP

Sodium azide (NaN3) Sigma (S-2002) 25 g

Sodium bicarbonate (NaHCO3) Sigma (S-4772) 1 kg

Sodium carbonate (Na2CO3) Sigma (S-1641) 1 kg

Sodium chloride (NaCl) Merck (1540) 500 g

Sodium hydroxide (NaOH) Sigma (S-8045) 500 g

Sodium phosphate (Na2HPO4) Fisher (S374-500) 500 g

2,3,5-triphenyl tetrazolium chloride Fisher (T-413) 10 g

Tri-sodium citrate (C6H5Na3O7.2H2O) Merck (6448.1000) 1 kg

Tween-20 Fisher (BP337-500) 500 ml

Vitamin C (tablets of 500 mg each) Pharmacy

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Code Name Chemical composition/packet or bottle

1 Coating buffer Na2CO3 = 0.159 gNaHCO3 = 0.293 gNaN3 = 0.02 g

2 PBS (phosphate buffer saline) NaCl = 8.0 gKH2PO4 = 0.2 gKCl = 0.2 gNa2HPO4 = 1.15 gNaN3 = 0.2 g

3 Tween-20 Tween-20 = 1 ml

4 Extraction buffer pH 7.4 NaCl = 16.0 g(PBS without sodium azide) KH2PO4 = 0 4 g

KCl = 0.4 gNa2HPO4 = 2.3 g

5 Conjugate buffer PVP-40 000 = 0.25 gNon-fat powdered milk = 0.025 g

6 Substrate buffer (5x) Diethanolamine = 10.9 mlHCl (37%) = 1.5 mlDistilled water = 6.0 ml

7 Immunoglobulins (IgG) Rs-specific rabbit IgG = 0.6 ml

8 Conjugated IgG Rs-specific rabbit IgG conjugatedto alkaline phosphatase = 0.6 ml

9 Substrate (p-np) p-nitrophenyl phosphate

10 Microtitration plates Polystyrene

11 Enrichment broth 10x Enrichment broth 10x (see Appendix 1)

12 Positive controls Boiled suspensions of Rs in citrate buffer(105–108 bacteria/ml)

13 Negative control Enriched extract of Rs-free soil

14 Citrate buffer pH 5.6 A = Citric acid (1.995 g)B = Tri-sodium citrate (11.907 g)

15 HCl HC1 (18.5%) = 6 ml

16 NaOH NaOH 1 N = 10 ml

LIST OF SUPPLIES

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Packaging Physical state Remarks

1 packet Powder, white Stable at 4°C

2 packets (each enough to make 1 liter of buffer) Powder, white Stable at 4°C

1 eppendorf tube Liquid, colorless Stable at 4°C

5 packets (each enough to make 2 liters of buffer) Powder, white Stable at 4°C

6 packets Powder, cream Prepare fresh for each test

1 bottle Liquid, colorless Stable at 4°C for 6 months

1 eppendorf tube Liquid, colorless Stable at 4ºC for 6 months

1 eppendorf tube Liquid, colorless Stable at 4°C for 6 months

1 packet of 6 tablets (5 mg each) Pellets, yellow Stable at 4ºC for 6 months

I packet of 6 plates Solid

5 bottles (50 ml each) Liquid, blue Stable at 4ºC for 6 months;maintain sterile

4 eppendorf tubes of 1 ml each Liquid, colorless

1 eppendorf tube Liquid, colorless

3 packets Solid, white Stable at 4ºC3 packets Solid, white Stable at 4ºC(each pair of packets (A+B) is enough tomake 1 liter of buffer)

1 bottle Liquid, colorless Avoid contact with skin

1 bottle Liquid, colorless Avoid contact with skin

INCLUDED IN THE KIT

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APPENDICES

Appendix 1. Preparation of the enrichment broth (1x)

To one liter of potato broth (prepared by boiling 200 g of potatoes in one liter of water forabout 10 minutes), add 10 g bacto peptone, 2.5 g dextrose and 1 g casamino acids. This isthe basal medium. Autoclave, then cool to 50°C and add the following filter-sterilized solutionsof antibiotics and vitamin C (concentrations in the final broth are given in parentheses):

• 10 ml of 1% polymyxin B sulphate (100 mg/liter)• 10 ml of 1% cycloheximide (100 mg/liter) or 5 ml of 10% benlate

(500 mg/liter)• 2.5 ml of 1% bacitracin (25 mg/liter)• 500 µl of 0.1% penicillin-G (0.5 mg/liter)• 500 µl of 1% chloramphenicol (5 mg/liter)• 500 µl of 1% crystal violet (5 mg/liter)• 5 ml of 1% 2,3,5- triphenyl tetrazolium chloride (TZC; 50 mg/liter)• 2.5 ml of a vitamin C solution prepared by dissolving a tablet containing 500 mg

vitamin C in 20 ml of distilled water (62.5 mg/liter)

Appendix 2. Protocol for sample collection

In fields planted with potato or other hosts, take samples from the plant rhizosphere. Infields already harvested (bare soil), samples should be taken at 20–30 cm depth to avoidareas of high moisture and temperature fluctuations that can influence bacterial populations.The soil moisture should be at its field capacity. Collect about 100 g of soil with a small handhoe. After each sample, disinfect the hoe with 0.5% sodium hypochloride and rinse withwater.

Collect the samples in polyethylene bags; keep the bags open until sampling is complete.Transport samples to the lab under conditions that avoid excess of heat. At the lab, open thebags immediately and store them in an aerated place to avoid the multiplication ofantagonistic and anaerobic bacteria. If possible, process the samples as soon as they arrive.If not, samples can be kept for up to a week if they are stored in a cool place (temperaturebetween 10 and 15°C). Maintain the moisture of soil samples at field capacity.

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Appendix 3. Sample size

Experimental field

To evaluate methods for controlling bacterial wilt in experimental units of 15–20 m2 in a soilhighly infested with Rs, take one soil sample of about 100 g from each 2 m2. Mix all thesamples well, then take two composite samples of 100 g of soil for each plot.

For a semi-quantification of the bacterial population in the soil make serial 10-fold dilutionsof the supernatant of the soil solution in citrate buffer up to 10–5 (10–1, 10–2, 10–3, 10–4 and10–5). These dilutions should be enriched by mixing 1 ml of each dilution with 9 ml ofenrichment broth and incubating them at 30°C for 48 hours under constant agitation.

The kit is sufficient for two evaluations of an experimental field of 12 plots of about15–20 m2 each

Seed productionSeed productionSeed productionSeed productionSeed production

For a qualitative evaluation of the inoculum in the soil (presence or absence of Rs) analyze50 samples per hectare. The samples should be taken in a randomized design following azig-zag or cross pattern. For each sample make two serial 10-fold dilutions of the supernatantof the soil solution in citrate buffer (10–1 and 10–2). These dilutions should be enriched bymixing 1 ml of each dilution with 9 ml of enrichment broth and incubating the mixture at30°C for 48 hours under constant agitation.

The kit is sufficient to evaluate two fields of approximately 1 ha each.

Please send your comments and feedback to:

Dr Sylvie PriouInternational Potato Center (CIP)Apartado 1558, Lima 12, Peru

Tel: +51 1 349 6017 Fax: +51 1 317 5326E-Mail: s.priou@cgiar.org

Edited by the Department of Training and Communications