ANNEX I TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF THE RING ROT BACTERIUM, CLAVIBACTER MICHIGANENSIS (Smith) Davis et al. ssp. SEPEDONICUS (Spieckermann et Kotthoff) Davis et al.
SCOPE OF THE TEST SCHEME

Appendix 6 Validated PCR protocol and reagents

1. Multiplex PCR protocol with internal PCR control (Pastrik, 2000)

1.3. PCR reaction conditions
Run the following programme:
1 cycle of:	(i)	3 minutes at 95 °C (denaturation of template DNA)
10 cycles of:	(ii)	1 minute at 95 °C (denaturation of template DNA)
	(iii)	1 minute at 64 °C (annealing of primers)
	(iv)	1 minute at 72 °C (extension of copy)
25 cycles of:	(v)	30 seconds at 95 °C (denaturation of template DNA)
	(vi)	30 seconds at 62 °C (annealing of primers)
	(vii)	1 minute at 72 °C (extension of copy)
1 cycle of:	(viii)	5 minutes at 72 °C (final extension)
	(ix)	hold at 4 °C.

Note:
This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) (iv), (v), (vi) and (vii) may be required for use with other models.