F1ANNEX IITEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
1.PCR protocol of Seal et al. (1993)
1.1.Oligonucleotide primers
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
Reagent | Quantity per reaction | Final concentration |
|---|---|---|
Sterile UPW | 17,65 µl | |
10X PCR buffer34 (15 mM MgCl2) | 2,5 µl | 1X (1,5 mM MgCl2) |
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)34 | 0,1 µl | 0,5 U |
Sample volume | 2,0 µl | |
Total volume | 25 µl | |
Method was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL. | ||
1.3.PCR reaction conditions
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.
1.4.Restriction enzyme analysis of amplicon.
PCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Ava II after incubation at 37 °C.
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..