F1ANNEX IITEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

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Appendix 4Buffers for test procedures

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:

Purpose

Quantity (per l)

Lubrol flakes

Deflocculant32

0,5 g

DC silicone antifoam

Anti-foam agent32

1,0 ml

Tetrasodium pyrophosphate

Anti-oxidant

1,0 g

Polyvinylpyrrolidone-40000 (PVP-40)

Binding of PCR inhibitors

50 g

For use with homogenisation extraction method

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.