Identification of the unculturable bacteria Candidatus arthromitus in the intestinal content of trouts using Dot blot and Southern blot techniques
2012
Cecchini, F. | Iacumin, L. | Fontanot, M. | Comi, G. | Manzano, M.
The rainbow trout gastroenteritis (RTGE) syndrome is affecting progressively more sites in various European countries causing significant economical losses for the rainbow trout industries. As Candidatus arthromitus, a not yet culturable bacteria, is the microorganism responsible for the RTGE, molecular methods have been identified as an important tool for the identification of this microorganism. An early, fast and specific detection of C. arthromitus using specific primers and probes, are important goals for the aquaculture sector. In the present study were used: a polymerase chain reaction (PCR), using universal primers annealing the 16S rRNA gene, to obtain amplification products representatives of the microbiota present in the intestinal content of trouts, including C. arthromitus when present; a denaturing gradient gel electrophoresis (DGGE) to produce DNA finger printings corresponding to the microbiota present in the intestinal contents of each trout analysed; a specific DNA probe designed within the 16S rDNA that was used both in Dot blot and Southern blot techniques to detect the unculturable bacterium C. arthromitus. Dot blot applied directly onto DNA extracted from the intestinal contents allowed a fast identification of the samples containing C. arthromitus. Southern blotting allowed the identification of the specific DNA band corresponding to C. arthromitus among the various DNA bands obtained by the utilisation of the universal primers and run in DGGE. Molecular methods such as Dot blot and Southern blot are useful methods for a fast identification of the unculturable C. arthromitus.
显示更多 [+] 显示较少 [-]