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Council Directive 93/85/EEC of 4 October 1993 on the control of potato ring rot
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When the FISH test is used as the first screening test and found to be positive, the IF test must be performed as a second compulsory screening test. When the FISH test 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.
Use validated C. m. subsp. sepedonicus -specific oligo-probes (Appendix 7). Preliminary testing with this method should permit reproducible detection of at least 10 3 to 10 4 cells of C. m. subsp. sepedonicus per ml added to sample extracts which previously tested negative.
The following procedure should preferably be performed on freshly prepared sample extract but can also be successfully performed on sample extract that has been stored under glycerol at -16 to -24 °C or -68 to -86 °C.
As negative controls, use aliquots of sample extract that previously tested negative for C. m. subsp. sepedonicus .
As positive controls prepare suspensions containing 10 5 to 10 6 cells per ml of C. m. subsp. sepedonicus (e.g. strain NCPPB 4053, or PD 406) in 0,01M phosphate buffer (PB) from a three to five day culture (preparation see Appendix 2). Prepare separate positive control slides of the homologous strain or any other reference strain of C. m. subsp. sepedonicus , suspended in potato extract, as specified in Appendix 2.
The use of the FITC-labelled eubacterial oligo-probe offers a control for the hybridisation process, since it will stain all eubacteria that are present in the sample.
Test control material in an identical manner as the sample(s).
The following protocol is based upon Wullings et al. , (1998): U.K.
An alternative fixative is 96 % ethanol. To use this dissolve the pellet from step 5.1.2 in 50 µl 0,01M PB and 50 µl 96 % ethanol. Vortex mix and incubate at 4 °C for 30 to 60 minutes.
At this stage the procedure may be interrupted and the hybridisation continued the following day. Slides should be stored dust-free and dry at room temperature.
Alternatively, instead of lysozyme add 50 µl of 40 to 400µg ml –1 proteinase K in buffer (20 mM Tris-HCl, 2 mM CaCl 2 , pH 7,4) to each well and incubate at 37 °C for 30 minutes.
Valid FISH test results are obtained if bright green fluorescent cells of size and morphology typical of C. m. subsp. sepedonicus are observed using the FITC filter and if bright red fluorescent cells using the rhodamine filter in all positive controls and not in any of the negative controls. 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 4). Samples with at least 5 × 10 3 typical cells per ml of resuspended pellet are considered potentially contaminated. Further testing is required. Samples with less than 5 × 10 3 typical cells per ml of resuspended pellet are considered negative.
The FISH test is negative if bright red fluorescent cells with size and morphology typical of C. m. subsp. sepedonicus are not observed using the rhodamine filter, provided that typical bright red fluorescent cells are observed in the positive control preparations when using the rhodamine filter.]
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