DETECTION OF RALSTONIA SOLANACEARUM RACE 3 IN POTATO TUBERS BY PCR AMPLIFICATION AND SANDWICH HYBRIDIZATION IN MICROTITRATION PLATES
A CHANDELIER1, S COGNET1 and P LEPOIVRE1
1Unité de Phytopathologie, FUSAGx, 2 Passage des Deportes, B-5030 Gembloux, Belgium
Background and objectives
From a practical point of view, there is a need for a sensitive test, able to detect the bacteria in latently infected tubers. Moreover, considering the great number of samples to analyse, it is also important to develop a test which can be automated. In that respect, we aim at developing a diagnostic kit based on PCR amplification of ribosomal DNA and detection of the amplicons by sandwich hybridization in microtitration plates.
Materials and methods
Results and conclusions
A sandwich hybridization assay in microtitration plate was then performed using the kit in development from Lambdatech s.a.. The amplified DNA was hybridized between a capture probe and three biotinylated detection probes. The hybrids were then detected with a streptavidin-peroxydase conjugate after the addition of the revelation mixture. In contrast to non-targeted bacteria, positive samples gave rise to high optical density values (ca 2.0), thus suggesting that the proposed assay can distinguish between targeted and non-targeted bacteria.
In order to characterize the sensitivity of our test, known concentrations of R. solanacearum race 3 were added to 1 ml of plant sap and total nucleic acids were extracted using the silica-based protocol. The detection limit after sandwich hybridization was about 10,000 bacteria/ml plant sap. As this detection limit is not sufficient, we aim in the future at improving the sample preparation step, either by modifying the silica-based protocol or by performing a pre-culture in a selective medium of the infected tubers (bioPCR).