DAS-ELISA kit for the detection of Ralstonia solanacearum in soil. Instructions for use by International Potato Center

DAS-ELISA kit For the detection of Ralstonia solanacearum in soil

INSTRUCTIONS FOR USE

INTERNATIONAL POTATO CENTER (CIP) 2001

DAS-ELISA kit For the detection of Ralstonia solanacearum in soil INSTRUCTIONS FOR USE Dr Sylvie Priou International Potato Center (CIP) 2001

Contents

Introduction

Page 5

Materials

Sample preparation

Serological test

Sources/suppliers of the products included in the kit

List of supplies included in the kit

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

18 18 19

Coat the wells of a microtitration plate with Ralstonia solanacearum (Rs)-specific rabbit immunoglobulins (IgG)

Microtitration plate Rs-specific bbit immunoglobulins (IgG) Rs in soil solution

Add soil solution; Rs in the soil will bind to the IgG

Enzyme-labeled Rsspecific rabbit IgG (conjugated IgG) Enzyme substrate

Add enzyme-labeled Rs-specific rabbit IgG; these will bind to the Rs-IgG complex to form the double antibody sandwich

Add the enzyme substrate; if Rs is present a color will develop

Figure 1: The four steps of the DAS-ELISA p Procedure

INTRODUCTION DAS-ELISA (double antibody sandwich enzyme-linked inmunosorbent assay) is an immunoenzymatic 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 rabbit immunoglobulins (IgG) 2. Adding solutions prepared from soil samples to the wells of the microtitration plate 3. 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 the DAS-ELISA an enrichment procedure must be carried out to allow the bacteria to multiply to detectable levels. This is done by incubating the soil solution in an enrichment broth at 30째C for 48 hours with constant agitation. By this procedure the method is capable of detecting original 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 immunoglobulins provided in the kit. Some saprophytic bacteria may cross-react with the antibodies produced against 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 crossreactions 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, to evaluate 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 that you have all materials ready. 5

MATERIALS As soon as you receive the kit, wrap it in a plastic bag and store it in a refrigerator at 4°C. The products are stable for six months. The supplies included in the kit are sufficient for testing six microtitration plates of approximately 40 samples each (for sample size see Appendix 3 on Page 19). The supplies included in the kit are listed on page 16. The following equipment and materials are also needed for the assay but are not included in the kit (everything must be very clean): 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 store buffers • 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

• • • •

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

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 after preparation. Store in a refrigerator. The first time you prepare a buffer solution measure its pH with a pH meter, and then add HCl (bottle #15) or NaOH (bottle #16) as necessary to achieve the desired pH. Write the volumes of HCl or NaOH added in your manual for faster future applications (if the same water 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 pH to 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 water while 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 citrate buffer 8

90 ml PBS buffer + 10 g soil Agitation 30 mi Settling 40 seconds SOIL EXTRACTION

SERIAL 10-FOLD DILUTIONS IN CITRATE BUFFER

Dilution 1/10

Dilution 1/10 10 -1

ENRICHMENT

...

Dilution 1/10

10 -2

10 -5

1 ml of diluted soil extract + 9 ml of CIP enrichment broth

...

Incubation at 30ยบ C for 48 hours under constant agitation

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

Enrichment procedure (48 hours) • Dilute 50 ml of the 10x enrichment broth (one bottle #11) with 450 ml of sterile distilled water • Dispense 9 ml of the diluted (1x) enrichment broth in each of five sterile 25- or 50-ml erlenmeyers • Add 1 ml of a different dilution of the soil solution to each erlenmeyer of enrichment broth • 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.

SEROLOGICAL TEST All volumes and quantities indicated (except for the phosphate buffer saline (PBS)) are for a test using one microtitration plate. For each plate you will need exactly 250 ml of PBS buffer. 1. Coating the microtitration plate with Rs-specific rabbit immunoglobulins (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 eppendorf tube #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; ensure that all wells are filled. Cover the plate with a piece of parafilm to prevent evaporation and incubate at 37°C for 4 hours. This process will allow immunoglobulins to adhere to the surface 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 and adjust pH to 7.4.

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. (Tween20 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 washing buffer 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 and controls can be easily identified. With a micropipetor, transfer 125 µl (0.125 ml) samples of one of the enriched soil solutions to each of two wells of the microtitration plate. Repeat this process for all the other enriched soil 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 negative control (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 completely colorless. 12

3.2. Preparation of the conjugate buffer (12.5 ml) During the last washing, dissolve the contents of one packet #5 (conjugate buffer) in 12.5 ml 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 alkaline phosphatase (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 to each 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 by washing 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-nitrophenyl phosphate) 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 development solution 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. 13

4.5. Interpretation of the results In positive samples, the enzymatic reaction gives rise to a yellow coloration, ranging in intensity 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 least concentrated control (105 bacteria/ml) should be light yellow. The color can be measured with a spectrophotometer at 405 nm wavelength. The samples are considered positive if the absorbance is three times as much as the average of the enriched soil solution free of Rs used as the negative control.

SOURCES/SUPPLIERS OF THE PRODUCTS INCLUDED IN THE KIT Product

Supplying company (and catalog number)

Bacitracin

Sigma (B-0125)

Minimum quantity supplied by company 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)

Citric acid

Sigma (C-1909)

500 g

Conjugate (Rs-IgG)

CIP

Crystal violet

Sigma (C-3886)

25 g

Cycloheximide

Sigma (C-7698)

Dextrose

Fisher (D16-500)

500 g

Diethanolamine

Fisher (D-45-500)

500 ml

Hydrochloric acid â&#x20AC;&#x201C; 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)

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 (C6H5Na O 2H2O)

Merck (6448.1000)

1 kg

Tween-20

Fisher (BP337-500)

500 ml

Vitamin C (tablets of 500 mg each)

Pharmacy

. 3 7

LIST OF SUPPLIES Code

Name

Chemical composition/packet or bottle

Coating buffer

Na2CO3 = 0.159 g NaHCO3 = 0.293 g NaN3 = 0.02 g

PBS (phosphate buffer saline)

NaCl = 8.0 g KH2PO4 = 0.2 g KCl = 0.2 g Na2HPO4 = 1.15 g NaN3 = 0.2 g

Tween-20

Tween-20 = 1 ml

Extraction buffer pH 7.4 (PBS without sodium azide)

NaCl = 16.0 g KH2PO4 = 0 4 g KCl = 0.4 g Na2HPO4 = 2.3 g

Conjugate buffer

PVP-40 000 = 0.25 g Non-fat powdered milk = 0.025 g

Substrate buffer (5x)

Diethanolamine = 10.9 ml HCl (37%) = 1.5 ml Distilled water = 6.0 ml

Immunoglobulins (IgG)

Rs-specific rabbit IgG = 0.6 ml

Conjugated IgG

Rs-specific rabbit IgG conjugated to alkaline phosphatase = 0.6 ml

Substrate (p-np)

p-nitrophenyl phosphate

Microtitration plates

Polystyrene

Enrichment broth 10x

Enrichment broth 10x (see Appendix 1)

Positive controls

Boiled suspensions of Rs in citrate buffer (105â&#x20AC;&#x201C;108 bacteria/ml)

Negative control

Enriched extract of Rs-free soil

Citrate buffer pH 5.6

A = Citric acid (1.995 g) B = Tri-sodium citrate (11.907 g)

HCl

HC1 (18.5%) = 6 ml

NaOH

NaOH 1 N = 10 ml 16

INCLUDED IN THE KIT 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

4 eppendorf tubes of 1 ml each

Liquid, colorless

1 eppendorf tube

Liquid, colorless

3 packets 3 packets (each pair of packets (A+B) is enough to make 1 liter of buffer)

Solid, white Solid, white

Stable at 4ºC Stable at 4ºC

1 bottle

Liquid, colorless

Avoid contact with skin

1 bottle

Liquid, colorless

Avoid contact with skin

Stable at 4ºC for 6 months; maintain sterile

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 for about 10 minutes), add 10 g bacto peptone, 2.5 g dextrose and 1 g casamino acids. This is the basal medium. Autoclave, then cool to 50°C and add the following filter-sterilized solutions of 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. In fields already harvested (bare soil), samples should be taken at 20–30 cm depth to avoid areas 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 hand hoe. After each sample, disinfect the hoe with 0.5% sodium hypochloride and rinse with water. 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 the bags immediately and store them in an aerated place to avoid the multiplication of antagonistic 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 (temperature between 10 and 15°C). Maintain the moisture of soil samples at field capacity. 18

Appendix 3. Sample size

Experimental field To evaluate methods for controlling bacterial wilt in experimental units of 15–20 m2 in a soil highly infested with Rs, take one soil sample of about 100 g from each 2 m2. Mix all the samples 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 dilutions of the supernatant of the soil solution in citrate buffer up to 10–5 (10–1, 10–2, 10–3, 10–4 and 10–5). These dilutions should be enriched by mixing 1 ml of each dilution with 9 ml of enrichment 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 about 15–20 m2 each

Seed production For a qualitative evaluation of the inoculum in the soil (presence or absence of Rs) analyze 50 samples per hectare. The samples should be taken in a randomized design following a zig-zag or cross pattern. For each sample make two serial 10-fold dilutions of the supernatant of the soil solution in citrate buffer (10–1 and 10–2). These dilutions should be enriched by mixing 1 ml of each dilution with 9 ml of enrichment broth and incubating the mixture at 30°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 Priou International Potato Center (CIP) Apartado 1558, Lima 12, Peru Tel: +51 1 349 6017 Fax: +51 1 317 5326 E-Mail: s.priou@cgiar.org Edited by the Department of Training and Communications