[F1ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

Textual Amendments

Appendix 4 Buffers for test procedures

General: Unopened sterilised buffers can be stored for up to one year U.K.

1. Buffers for extraction procedure U.K.

1.1. Extraction buffer (50 mM phosphate buffer, pH 7,0) U.K.

This buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.

Na 2 HPO 4 (anhydrous) 4,26 g
KH 2 PO 4 2,72 g
Distilled water 1.00 l

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.

Additional components may be useful as follows:

a

For use with homogenisation extraction method

Purpose Quantity (per l)
Lubrol flakes Deflocculant a 0,5 g
DC silicone antifoam Anti-foam agent a 1,0 ml
Tetrasodium pyrophosphate Anti-oxidant 1,0 g
Polyvinylpyrrolidone-40000 (PVP-40) Binding of PCR inhibitors 50 g
1.2. Pellet buffer (10 mM phosphate buffer, pH 7,2) U.K.

This buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.

Na 2 HPO 4 .12H 2 O 2,7 g
NaH 2 PO 4 .2H 2 O 0,4 g
Distilled water 1,0 l

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

2. Buffers for the IF test U.K.

2.1. IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7.2) U.K.

This buffer is used for dilution of antibodies

Na 2 HPO 4 .12H 2 O 2,7 g
NaH 2 PO 4 .2H 2 O 0,4 g
NaCL 8,0 g
Distilled water 1,0 l

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

2.2. IF-buffer-Tween U.K.

This buffer is used to wash slides.

Add 0,1 % Tween 20 to the IF buffer.

2.3. Phosphate buffered glycerol, pH 7,6 U.K.

This buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.

Na 2 HPO 4 .12H 2 O 3,2 g
NaH 2 PO 4 .2H 2 O 0,15 g
Glycerol 50 ml
Distilled water 100 ml

Anti-fading mountant solutions are commercially available e.g. Vectashield ® (Vector Laboratories) or Citifluor ® (Leica).

3. Buffers for the Indirect ELISA test U.K.

3.1. Double strength coating buffer, pH 9,6. U.K.
Na 2 CO 3 6,36 g
NaHCO 3 11,72 g
Distilled water 1,00 l

Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 minutes.

Sodium sulphite (0.2 %) may be added as antioxidant if required to prevent build up of oxidised aromatic compounds.

3.2. 10X Phosphate buffered saline (PBS), pH 7,4 U.K.
NaCL 80,0 g
KH 2 PO 4 2,0 g
Na 2 HPO 4 .12H 2 O 29,0 g
KCl 2,0 g
Distilled water 1,0 L
3.3. PBS-Tween U.K.
10X PBS 100 ml
10 % Tween 20 5 ml
Distilled water 895 ml
3.4. Blocking (antibody) buffer (must be freshly prepared). U.K.
10X PBS 10,0 ml
Polyvinylpyrrolidone-44000 (PVP-44) 2,0 g
10 % Tween 20 0,5 ml
Milk powder 0,5 g
Distilled water make up to 100 ml
3.5. Alkaline phosphatase substrate solution, pH 9,8 U.K.
Diethanolamine 97 ml
Distilled water 800 ml

Mix and adjust to pH 9,8 with concentrated HCl.

Make up to 1 L with distilled water.

Add 0,2 g MgCl 2 .

Dissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.

4. Buffers for DASI ELISA test U.K.

4.1. Coating buffer, pH 9,6 U.K.
Na 2 CO 3 1,59 g
NaHCO 3 2,93 g
Distilled water 1 000  ml

Dissolve ingredients and check pH 9,6

4.2. 10X Phosphate saline buffer(PBS) pH 7,2 to 7,4 U.K.
NaCl 80,0 g
NaH 2 PO 4 .2 H 2 O 4,0 g
Na 2 HPO 4 .12H 2 O 27,0 g
Distilled water 1 000  ml
4.3. PBS-Tween U.K.
10X PBS 50 ml
10 % Tween 20 5 ml
Distilled water 950 ml
4.4. Substrate buffer, pH 9,8 U.K.
Diethanolamine 100 ml
Distilled water 900 ml

Mix and adjust to pH 9,8 with concentrated HCl.]