[F1ANNEX II U.K. TEST SCHEME FOR DIAGNOSIS, DETECTION AND IDENTIFICATION OF RALSTONIA SOLANACEARUM (SMITH) YABUUCHI ET AL.
Textual Amendments
SECTION VI U.K. OPTIMISED PROTOCOLS FOR DETECTION AND IDENTIFICATION OF R. SOLANACEARUM
A. DIAGNOSTIC AND DETECTION TESTS U.K.
5. IF Test U.K.
Principle 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).
5.1. Prepare the test slides by one of the following procedures: U.K.
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.
5.2. Dry the droplets at ambient temperature or by warming to temperatures of 40 to 45 °C. Fix the bacterial cells to the slide either by heating (15 minutes at 60 °C), flaming, with 95 % ethanol or according to specific instructions from the suppliers of the antibodies. U.K.
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.
5.3. IF procedure U.K.
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.
Figure 1.
Preparation of the test slide according to 5.1(i) and 5.3(i)
| Dilutions of resuspended pellet | |||||||
| 1/100 | 1/1 | 1/1 | 1/1 | 1/1 | | Dilution of resuspended pellet | |
| (T = titre) | T/2 | T/4 | T/2 | T | 2T | | Twofold dilutions of antiserum/antibody |
| Sample 1 |  |  |  |  |  | ||
| 1 | 2 | 3 | 4 | 5 | |||
| Duplicate of sample1 or sample 2 |  |  |  |  |  | ||
| 6 | 7 | 8 | 9 | 10 | |||
Figure 2.
Preparation of the test slide according to 5.1(ii) and 5.3(ii).
| Working dilution of antiserum/antibody | |||||||
| 1/1 | 1/10 | 1/100 | empty | empty | | Decimal dilution of resuspended pellet | |
| Sample 1 |  |  |  |  |  | ||
| 1 | 2 | 3 | 4 | 5 | |||
| Duplicate of sample 1 or sample 2 |  |  |  |  |  | ||
| 6 | 7 | 8 | 9 | 10 | |||
5.3.1. Arrange the slides on moist tissue paper. Cover each test window completely with the antibody dilution(s). The volume of antibody applied on each window must be at least the volume of extract applied. U.K.
The following procedure should be carried out in the absence of specific instructions from the suppliers of the antibodies:
5.3.2. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C). U.K.
5.3.3. Shake the droplets off each slide and rinse carefully with IF buffer. Wash by submerging for five minutes in IF buffer-Tween (Appendix 4) and subsequently in IF buffer. Avoid causing aerosols or droplet transfer that could result in cross-contamination. Carefully remove excess moisture by blotting gently. U.K.
5.3.4. Arrange the slides on moist paper. Cover the test windows with the dilution of FITC conjugate used to determine the titre. The volume of conjugate applied on the windows must be identical to the volume of antibody applied. U.K.
5.3.5. Incubate the slides on moist paper under a cover for 30 minutes at ambient temperature (18 to 25 °C). U.K.
5.3.6. Shake the droplets of conjugate off the slide. Rinse and wash as before (5.3.3). U.K.
Carefully remove excess moisture.
5.3.7. Pipette 5 - 10 µl of 0,1M phosphate-buffered glycerol (Appendix 4) or a commercially antifading mountant on each window and apply a coverslip. U.K.
5.4. Reading the IF test: U.K.
5.4.1 Examine test slides on an epifluorescence microscope with filters suitable for excitation of FITC, under oil or water immersion at a magnification of 500- 1 000 . Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields. U.K.
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.
5.4.2. Observe for bright fluorescing cells with characteristic morphology of R. solanacearum in the test windows of the test slides (see website http://forum.europa.eu.int/Public/irc/sanco/Home/main). The fluorescence intensity must be equivalent to the positive control strain at the same antibody dilution. Cells with incomplete staining or with weak fluorescence must be disregarded. U.K.
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.
5.4.3. There are several problems inherent to the specificity of the immunofluorescence test. Background populations of fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology similar to R. solanacearum are likely to occur in potato heel end core and stem segment pellets. U.K.
5.4.4. Consider only fluorescing cells with typical size and morphology at the titre or working dilution of the antibodies as in 5.3. U.K.
5.4.5. Interpretation of the IF reading: U.K.
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.]