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Textual Amendments
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).
For pellets with relatively little starch sediment:
Pipette a measured standard volume (15 µl is appropriate for 6 mm window diameter – scale up volume for larger windows) of a 1/100 dilution of the resuspended potato pellet onto the first window. Subsequently pipette a similar volume of undiluted pellet (1/1) onto the remaining windows on the row. The second row can be used as duplicate or for a second sample as presented in Figure 1.
For other pellets:
Prepare decimal dilutions (1/10, 1/100) of the resuspended pellet in pellet buffer. Pipette a measured standard volume (15 µl is appropriate for 6 mm window diameter – scale up volume for larger windows) of the resuspended pellet and each dilution on a row of windows. The second row can be used as duplicate or for a second sample as presented in Figure 2.
If necessary, fixed slides may then be stored frozen in a desiccated box for as little time as necessary (up to a maximum of three months) prior to further testing.
According to test slide preparation in 5.1(i):
Prepare a set of twofold dilutions The first well should have 1/2 of the titre (T/2), the others 1/4 of the titre (T/4), 1/2 of the titre (T/2), the titre (T) and twice the titre (2T).
According to test slide preparation in 5.1(ii):
Prepare the working dilution (WD) of the antibody in IF buffer. The working dilution affects the specificity.
The following procedure should be carried out in the absence of specific instructions from the suppliers of the antibodies:
Carefully remove excess moisture.
Check the positive control slide first. Cells must be bright fluorescent and completely stained at the determined antibody titre or working dilution. The IF test (Section VI.A.5.) must be repeated if the staining is aberrant.
If any contamination is suspected the test must be repeated. This may be the case when all slides in a batch show positive cells due to the contamination of buffer or if positive cells are found (outside of the slide windows) on the slide coating.
If bright fluorescing cells with characteristic morphology are found, estimate the average number of typical cells per microscope field and calculate the number of typical cells per ml of resuspended pellet (Appendix 5).
The IF reading is positive for samples with at least 5 × 10 3 typical cells per ml of resuspended pellet. The sample is considered potentially contaminated and further testing is required.
The IF reading is negative for samples with less than 5 × 10 3 cells per ml of resuspended pellet and the sample is considered negative. Further testing is not required.]