Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al. (c. 57)

[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..

SECTION VI U.K. OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM

A. DIAGNOSTIC AND DETECTION TESTS U.K.

6. PCR tests U.K.

Principles U.K.

When the PCR test is used as the principal screening test and found to be positive, isolation or IF must be performed as a second compulsory screening test. When PCR is used as the second screening test and found to be positive, further testing according to the flow scheme is required to complete the diagnosis.

Full exploitation of this method as principal screening test is only recommended when specialised expertise has been acquired.

Note: Preliminary testing with this method should permit reproducible detection of 10 ³  to 10 ⁴ cells of R. solanacearum per ml added to sample extracts which previously tested negative. Optimisation experiments may be required to achieve maximum levels of sensitivity and specificity in all laboratories. U.K.

Use validated PCR reagents and protocols (see Appendix 6). Preferably select a method with an internal control.

Use appropriate precautions to avoid contamination of sample with target DNA. The PCR test should be performed by experienced technicians, in dedicated molecular biology laboratories, in order to minimise the possibility of contamination with target DNA.

Negative controls (for DNA extraction and PCR procedures) should always be handled as final samples in the procedure, to make evident whether any carry over of DNA has occurred.

The following negative controls should be included in the PCR test:

• Sample extract that previously tested negative for R. solanacearum ,

• Buffer controls used for extracting the bacterium and the DNA from the sample,

• PCR-reaction mix.

The following positive controls should be included:

• Aliquots of resuspended pellets to which R. solanacearum has been added (preparation see Appendix 3 B).

• A suspension of 10 ⁶ cells per ml of R. solanacearum in water from a virulent isolate (e.g. NCPPB 4156 = PD 2762 = CFBP 3857; see Appendix 3 B).

• If possible use also DNA extracted from positive control samples in the PCR test.

To avoid potential contamination prepare positive controls in a separate environment from samples to be tested.

Sample extracts should be as free as possible from soil. It could therefore, in certain cases, be advisible to prepare extracts from washed potatoes if PCR protocols are to be used.

Standardized positive and negative control material available for use with this test are listed in Appendix 3).]