[F1B. Preparation of positive and negative controls for the core screening tests PCR/IF and FISH U.K.
Produce a 48 hour culture of a virulent strain of R. solanacearum race3/biovar2 (e.g. strain NCPPB 4156 = PD 2762 = CFBP 3857) on basal SMSA medium and suspend in 10 mM phosphate buffer to obtain a cell density of approximately 2 × 10 8 cfu per ml. This is usually obtained by a faintly turbid suspension equivalent to an optical density of 0,15 at 600 nm.
Remove the heel end cores of 200 tubers taken from a white skin variety production known to be free from R. solanacearum .
Process the heel ends as usual and resuspend the pellet in 10 ml.
Prepare 10 sterile 1,5 ml microvials with 900 µl of the resuspended pellet.
Transfer 100 µl of the suspension of R. solanacearum to the first microvial. Vortex.
Establish decimal levels of contamination by further diluting in the next five microvials.
The six contaminated microvials will be used as positive controls. The four non-contaminated microvials will be used as negative controls. Label the microvials accordingly.
Prepare aliquots of 100 µl in sterile 1,5 ml microvials thus obtaining nine replicas of each control sample. Store at -16 to -24 °C until use.
The presence and quantification of R. solanacearum in the control samples should be first confirmed by IF.
For the PCR test perform DNA extraction from positive and negative control samples with each series of test samples.
For IF and FISH tests perform assays on positive and negative control samples with each series of test samples.
For IF, FISH and PCR assays R. solanacearum must be detected in at least the 10 6 and 10 4 cells/ml of the positive controls and not in any of the negative controls.]