F1ANNEX IITEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
General: Unopened sterilised buffers can be stored for up to one year
1.Buffers for extraction procedure
1.1.Extraction buffer (50 mM phosphate buffer, pH 7,0)
This buffer is used for extraction of the bacterium from plant tissues by homogenisation or shaking.
Na2HPO4 (anhydrous) | 4,26 g |
KH2PO4 | 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:
1.2.Pellet buffer (10 mM phosphate buffer, pH 7,2)
This buffer is used for resuspension and dilution of potato tuber heel-end core extracts following concentration to a pellet by centrifugation.
Na2HPO4.12H2O | 2,7 g |
NaH2PO4.2H2O | 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
2.1.IF-Buffer (10 mM phosphate buffered saline (PBS), pH 7.2)
This buffer is used for dilution of antibodies
Na2HPO4.12H2O | 2,7 g |
NaH2PO4.2H2O | 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
This buffer is used to wash slides.
Add 0,1 % Tween 20 to the IF buffer.
2.3.Phosphate buffered glycerol, pH 7,6
This buffer is used as a mountant fluid on the windows of IF slides to enhance fluorescence.
Na2HPO4.12H2O | 3,2 g |
NaH2PO4.2H2O | 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
3.1.Double strength coating buffer, pH 9,6.
Na2CO3 | 6,36 g |
NaHCO3 | 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
NaCL | 80,0 g |
KH2PO4 | 2,0 g |
Na2HPO4.12H2O | 29,0 g |
KCl | 2,0 g |
Distilled water | 1,0 L |
3.3.PBS-Tween
10X PBS | 100 ml |
10 % Tween 20 | 5 ml |
Distilled water | 895 ml |
3.4.Blocking (antibody) buffer (must be freshly prepared).
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
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 MgCl2.
Dissolve 2 phosphatase substrate 5 mg tablets (Sigma) per 15 ml of solution.
4.Buffers for DASI ELISA test
4.1.Coating buffer, pH 9,6
Na2CO3 | 1,59 g |
NaHCO3 | 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
NaCl | 80,0 g |
NaH2PO4.2 H2O | 4,0 g |
Na2HPO4.12H2O | 27,0 g |
Distilled water | 1 000 ml |
4.3.PBS-Tween
10X PBS | 50 ml |
10 % Tween 20 | 5 ml |
Distilled water | 950 ml |
4.4.Substrate buffer, pH 9,8
Diethanolamine | 100 ml |
Distilled water | 900 ml |
Mix and adjust to pH 9,8 with concentrated HCl.
Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al..