[F1Principle U.K.
The use of the IF test as the principal screening test is recommended because of its proven robustness to achieve the required thresholds.
When the IF test is used as the principal screening test and the IF reading is positive, the Isolation, PCR or FISH test must be performed as a second screening test. When the IF test is used as the second screening test and the IF reading is positive, further testing according to the flow scheme is required to complete the analysis.
Note: Use a 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 an appropriate fluorescein isothiocyanate (FITC) conjugate according to the manufacturer’s recommendations. Validated polyclonal antisera all had an IF titre of at least 1: 2 000 . During testing, the antibodies should be used at a working dilution(s) close to or at the titre. U.K.
The test should be performed on freshly-prepared sample extracts. If necessary, it can be successfully performed on extracts stored at -68 to -86 °C under glycerol. Glycerol can be removed from the sample by addition of 1 ml pellet buffer (Appendix 4), re-centrifugation for 15 minutes at 7 000 g and re-suspension in an equal volume of pellet buffer. This is often not necessary, especially if samples are fixed to the slides by flaming.
Prepare separate positive control slides of the homologous strain or any other reference strain of R. solanacearum , suspended in potato extract, as specified in Appendix 3 B, and optionally in buffer.
Naturally infected tissue (maintained by lyophilisation or freezing at -16 to -24 °C) should be used where possible as a similar control on the same slide.
As negative controls, aliquots of sample extract which previously tested negative for R. solanacearum can be used.
Standardised positive and negative control materials available for use with this test are listed in Appendix 3.
Use multiwell microscope slides with preferably 10 windows of at least 6 mm diameter.
Test control material in an identical manner as the sample(s).]