Prsentation PowerPoint

1
Anne-Claire Le Roux
aleroux_at_rennes.inra.fr
Detection of Ralstonia solanacearum and
Clavibacter michiganensis sepedonicus bacteria
INRA UMR BiO3P Rennes, 35653 Le Rheu cedex
FNPPPT, 9 rue dAthènes, 75009 Paris GNIS, 44
rue du Louvre F- 75 001, Paris
Ralstonia solanacearum (RS) and Clavibacter
michiganensis sepedonicus (CMS) are quarantine
bacteria - that is, they are harmful organisms
which may have economic importance in specific
areas. No tolerance for these organisms is
accepted it is therefore necessary to avoid
introducing them via international trade, or
allowing their spread across an area. Combating
them depends essentially on preventative methods
and in particular early detection of the
bacterium in various environments which may
harbour it, such as seed potatoes, water, soil or
adventitious shoots.
? To evaluate and optimise existing detection
tools ? To develop new fast, sensitive and
specific tools ? To monitor new technical
advances
Serological tools
? PRODUCTION OF POLYCLONAL ANTISERA
Characteristics of some antisera
Diagram of antisera production
Evaluation of antisera Determination of the
Titre, Dilution in use and Specificity
Antigen suspension (dead bact.)
Serums
Conservation at -20C
3 to 6 injections
? 14 RS antisera produced in 1996 ? 4 kept after
evaluation ? 6 CMS antisera produced in 1999 ? 5
kept ? 6 RS antisera produced in 2000 ? 5 kept
? These antisera are used for Immunofluorescence,
a technique recommended by the EU for detecting
quarantine bacteria
? PRIMERS SYNTHESIS
RDNA sequence 16S
? Three primers have been defined using
nucleotides difference in position 458-460 on DNA
16S, two specific to division 2 (D2 et Z) and one
to division 1 (D1) of RS.
? These primers have a good sensitivity and
specificity
? MULTIPLEX PCR
? A technique that detects both RS and CMS
bacteria simultaneously in a single PCR, which
saves time
? Definition of the different parameters of the
technique
? Comparison of sensitivities obtained by
conventional PCR (A) and multiplex PCR (B) on
ranges of DNA dilutions
? Same sensitivity from both techniques and both
DNAs 1.5 pg
MT 1 2 3 4 5 6 7 8 9
? Sensitivity of multiplex PCR on potato
macerates artificially contaminated by bacterial
suspensions of RS and CMS
215 pb (CMS)
? Detection in the order of 2 to 20 cfu per PCR
reaction
? These tools are transferred to various
seed-potato health monitoring laboratories during
training courses.
? These tools are also used as part of
epidemiological studies carried out on various
environments plant, soil, water.
? Extensive work in technological monitoring and
optimisation of techniques is done so as to
always have the best tools.
? Research into techniques to quantify the
inoculum by PCR is proposed.
Manager INRA Françoise Montfort
November 2003