Council Directive 98/57/ECShow full title

[F1 Appendix 6 Validated PCR protocols and reagents

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..

NB: Preliminary testing should permit reproducible detection of 10 ³ to 10 ⁴ cells of R. solanacearum per ml of sample extract. U.K.

Preliminary testing should also show no false positive results with a panel of selected bacterial strains (see Appendix 3).

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.

2. PCR protocol of Pastrik and Maiss (2000) U.K.

2.1. Oligonucleotide primers U.K.

Forward primer Ps-1	5′- agt cga acg gca gcg ggg g -3′
Reverse primer Ps-2	5′- ggg gat ttc aca tcg gtc ttg ca -3′

Expected amplicon size from R. solanacearum template DNA = 553 bp.

2.2. PCR reaction mix U.K.

a Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.
N.B. Originally optimised for MJ Research PTC 200 thermocycler with Gibco Taq Polymerase. Perkin Elmer AmpliTaq and buffer can also be used at the same concentrations.
Reagent	Quantity per reaction	Final concentration
Sterile UPW	16,025 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 (20 mM)	0,125 µl	0,1 mM
Primer Ps-1 (10 µM)	0,5 µl	0,2 µM
Primer Ps-2 (10 µM)	0,5 µl	0,2 µM
Taq polymerase (5U/µl) a	0,1 µl	0,5 U
Sample volume	5,0 µl
Total volume:	25,0 µl

2.3. PCR reaction conditions U.K.

Run the following programme:

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 68 °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 is 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.

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

PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Taq I after incubation at 65 °C for 30 minutes. The restriction fragments obtained from R. solanacearum -specific fragment are 457 bp and 96 bp in size.

3. Multiplex PCR protocol with internal PCR control (Pastrik et al. , 2002) U.K.

3.1. 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′
Forward primer NS-5-F	5′- AAC TTA AAG GAA TTG ACG GAA G -3′
Reverse primer NS-6-R	5′- GCA TCA CAG ACC TGT TAT TGC CTC -3′

Expected amplicon size from R. solanacearum template DNA = 718 bp (RS-primer set)

Expected amplicon size from the 18S rRNA internal PCR control = 310 bp (NS-primer set).

3.2. PCR reaction mix U.K.

a Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.
b Concentration of primers NS-5-F and NS-6-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see Section VI.A.6.1.a.). Re-optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation methods are used.
Reagent	Quantity per reaction	Final concentration
Sterile UPW	12,625 µl
10X PCR buffer a (15 mM MgCl 2 )	2,5 µl	1X (1,5 mM MgCl 2 )
BSA (fraction V) (10 %)	0,25 µl	0,1 %
d-nTP mix (20 mM)	0,125 µl	0,1 mM
Primer RS-1-F (10 µM)	2,0 µl	0,8 µM
Primer RS-1-R (10 µM)	2,0 µl	0,8 µM
Primer NS-5-F (10 µM) b	0,15 µl	0,06 µM
Primer NS-6-R (10 µM) b	0,15 µl	0,06 µM
Taq polymerase (5 U/µl) a	0,2 µl	1,0 U
Sample volume	5,0 µl
Total volume:	25,0 µl

3.3. PCR reaction conditions U.K.

Run the following programme:

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 is 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.

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

PCR products amplified from R. solanacearum DNA 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.

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.

5. Preparation of the loading buffer U.K.

5.1. Bromphenol blue (10 %- stock solution ) U.K.

Bromphenol blue	5 g
Distilled water (bidest)	50 ml

5.2. Loading buffer U.K.

Glycerol (86 %)	3,5 ml
Bromphenol blue (5,1)	300 µl
Distilled Water (bidest)	6,2 ml

6. 10X Tris Acetate EDTA (TAE) buffer, pH 8.0 U.K.

Tris buffer	48,40 g
Glacial acetic acid	11,42 ml
EDTA (disodium salt)	3,72 g
Distilled water	1,00 L

Dilute to 1X before use.

Also commercially available (e.g. Invitrogen or equivalent).]