Division

F1Note: Use an validated source of antibodies to R. solanacearum (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main) It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 10 ⁵ to 10 ⁶ cells per ml of the homologous strain of R. solanacearum and using appropriate secondary antibody conjugates according to the manufacturer’s recommendations. During testing, the antibodies should be used at a working dilution close to or at the titre of the commercial formulation.

Determine the titre of the antibodies on a suspension of 10 ⁵ to 10 ⁶ cells per ml of the homologous strain of R. solanacearum .

Include a sample extract that previously tested negative for R. solanacearum and a suspension of a non-cross reacting bacterium in phosphate buffered saline (PBS) as negative controls.

As positive control use aliquots of sample extract, that previously tested negative, mixed with 10 ³ to 10 ⁴ cells per ml of R. solanacearum biovar 2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857, see Appendix 2 A and B). For comparison of results on each plate use a standard suspension of 10 ⁵ to 10 ⁶ cells per ml in PBS of R. solanacearum . Ensure positive controls are well separated on the microtitre plate from the sample(s) under test.

Standardised positive and negative control materials available for use with this test are listed in Appendix 3 A.

Test control material in an identical manner as the sample(s).