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

Textual Amendments

Appendix 6 Validated PCR protocols and reagents

1. PCR protocol of Seal et al. (1993) U.K.

1.1. Oligonucleotide primers U.K.
Forward primer OLI-1 5′-GGG GGT AGC TTG CTA CCT GCC-3′
Reverse primer Y-2 5′-CCC ACT GCT GCC TCC CGT AGG AGT-3′

Expected amplicon size from R. solanacearum template DNA = 288 bp

1.2. PCR reaction mix U.K.
a

Method was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.

Reagent Quantity per reaction Final concentration
Sterile UPW 17,65 µl
10X PCR buffer a (15 mM MgCl 2 ) 2,5 µl 1X (1,5 mM MgCl 2 )
dNTP mix (20 mM) 0,25 µl 0,2 mM
Primer OLI-1 (20 µM) 1,25 µl 1µM
Primer Y-2 (20 µM) 1,25 µl 1µM
Taq polymerase (5U/µl) a 0,1 µl 0,5 U
Sample volume 2,0 µl
Total volume 25 µl
1.3. PCR reaction conditions U.K.

Run the following programme:

1 cycle of: (i) 2 minutes at 96 °C (denaturation of template DNA)
35 cycles of: (ii) 20 seconds at 94 °C (denaturation of template DNA)
(iii) 20 seconds at 68 °C (annealing of primers)
(iv) 30 seconds at 72 °C (extension of copy)
1 cycle of: (v) 10 minutes at 72 °C (final extension)
(vi) hold at 4 °C.

NB: This programme was optimised for use with a Perkin Elmer 9600 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models. U.K.

1.4. Restriction enzyme analysis of amplicon. U.K.

PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Ava II after incubation at 37 °C.]