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[F1 ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.

Textual Amendments

F1 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..

Appendix 6 Validated PCR protocols and reagents

4. R. solanacearum biovar-specific PCR protocol (Pastrik et al. , 2001) U.K.

4.1. Oligonucleotide primers U.K.

Forward primer Rs-1-F	5′- ACT AAC GAA GCA GAG ATG CAT TA -3′
Reverse primer Rs-1-R	5′- CCC AGT CAC GGC AGA GAC T -3′
Reverse primer Rs-3-R	5′- TTC ACG GCA AGA TCG CTC -3′

Expected amplicon size from R. solanacearum template DNA:

with Rs-1-F/Rs-1-R = 718 bp

with Rs-1-F/Rs-3-R = 716 bp.

4.2. PCR reaction mix U.K.

(a)

Biovar 1/2-specific PCR

a Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.
Reagent	Quantity per reaction	Final concentration
Sterile UPW	12,925 µl
10X PCR Buffer a	2,5 µl	1X (1,5 mM MgCl 2 )
BSA (fraction V) (10 %)	0,25 µl	0,1 %
d-NTP mix (20mM)	0,125 µl	0,1 mM
Primer Rs-1-F (10 µM)	2 µl	0,8 µM
Primer Rs-1-R (10 µM)	2 µl	0,8 µM
Taq polymerase (5U/µl) a	0,2 µl	1 U
Sample volume	5,0 µl
Total volume	25,0 µl

(b)

Biovar 3/4/5-specific PCR

a Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.
Reagent	Quantity per reaction	Final concentration
Sterile UPW	14,925 µl
10X PCR Buffer a	2,5 µl	1X (1,5 mM MgCl 2 )
BSA (fraction V) (10 %)	0,25 µl	0,1 %
dNTP mix (20 mM)	0,125 µl	0,1 mM
Primer Rs-1-F (10 µM)	1 µl	0,4 µM
Primer Rs-3-R (10 µM)	1 µl	0,4 µM
Taq polymerase (5 U/µl) a	0,2 µl	1 U
Sample volume	5,0 µl
Total volume	25,0 µl

4.3. PCR reaction conditions U.K.

Run the following programme for both biovar 1/2- and biovar 3/4/5-specific reactions:

1 cycle of:	(i)	5 minutes at 95 °C (denaturation of template DNA)
35 cycles of:	(ii)	30 seconds at 95 °C (denaturation of template DNA)
	(iii)	30 seconds at 58 °C (annealing of primers)
	(iv)	45 seconds at 72 °C (extension of copy)
1 cycle of:	(v)	5 minutes at 72 °C (final extension)
	(vi)	hold at 4 °C.

NB: This programme was optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models. U.K.

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

PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-1-R produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes. PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-3-R have no restriction sites.]