F1ANNEX ITEST 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
1.Oligo-probes
Cms-specific probe CMS-CY3-01: | 5′- ttg cgg ggc gca cat ctc tgc acg -3′ |
Non-specific eubacterial probe EUB-338-FITC: | 5′- gct gcc tcc cgt agg agt -3′ |
2.Fixative solution
[WARNING! THE FIXATIVE CONTAINS PARAFORMALDEHYDE WHICH IS TOXIC. WEAR GLOVES AND DO NOT INHALE. IT IS ADVISABLE TO WORK IN A FUME CUPBOARD]
- (i)
Heat nine ml molecular grade water (e.g. Ultra pure water (UPW)) to about 60 °C and add 0,4 g paraformaldehyde. Paraformaldehyde dissolves after adding five drops of 1N NaOH and stirring with a magnetic stirrer.
- (ii)
Adjust pH to 7,0 by addition of 1ml of 0,1 M phosphate buffer (PB; pH 7,0) and five drops of 1 N HCl. Check pH with indicator strips and adjust if necessary with HCl or NaOH.
[WARNING! DO NOT USE A PH METER IN SOLUTIONS WITH PARAFORMALEDHYDE]
- (iii)
Filter the solution through a 0,22 µm membrane filter and keep dust-free at 4 °C until further use.
- (iv)
Note:
Alternative fixative solution: 96 % ethanol.
3.3x Hybmix
NaCl | 2,7 M |
Tris-HCl | 60 mM (pH 7,4) |
EDTA (filter sterilised and autoclaved) | 15 mM |
Dilute to 1x as required.
4.Hybridisation solution
1x Hybmix
Sodium dodecyl sulphate (SDS) | 0,01 % |
probe EUB 338 | 5 ng/μl |
probe CMSCY301 | 5 ng/μl |
Prepare quantities of hybridisation solution according to the calculations in Table. For each slide (containing two different samples in duplicate) 90 μl hybridisation solution is required.
2 slides | 8 slides | |
|---|---|---|
Sterile UPW | 50,1 | 200,4 |
3x hybmix | 30,0 | 120,0 |
1 % SDS | 0,9 | 3,6 |
Probe EUB 338 (100 ng/μl) | 4,5 | 18,0 |
Probe CMSCY301 (100 ng/μl) | 4,5 | 18,0 |
Total volume (μl) | 90,0 | 360,0 |
NB. Store all solutions containing light sensitive oligo-probes in the dark at -20 °C. Protect from direct sunlight or electric light during use.
5.0,1M Phosphate buffer, pH 7,0
Na2HPO4 | 8,52 g |
KH2PO4 | 5,44 g |
Distilled water | 1,00 L |
Dissolve ingredients, check pH and sterilise by autoclaving at 121 °C for 15 min.
Substituted by Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot.