Tipificación molecular de brucella y aplicación de la pcr al diagnóstico de la brucelosis

Resum

Molecular typing of Brucella y application of PCR for the diagnosis of brucellosis. David García Yoldi. Faculty of Pharmacy, University of Navarra (Spain), 2009. Brucella causes brucellosis, one of the most important bacterial zoonosis in the world. The main objective was develop and evaluate different PCR-based techniques for the detection and molecular typing of Brucella at species, biovar and strains levels. Different PCR-based techniques have been evaluated: conventional PCR, multiplex PCR, real time PCR, PCR-RFLP and MLVA (multiple-locus variable-number tandem repeat analysis). Genomic sequences of different Brucella strains were analysed and conserved sequences were selected for the detection of genus Brucella. In addition, based on the analysis of genetic polymorphisms among Brucella species and biovars several molecular markers were selected (including insertions, deletions, punctual mutations and tandem repeats). A new multiplex PCR assay (named Bruce-ladder) was developed. This new test was specific for Brucella and can identify and differentiate all the Brucella species and the vaccine strains. Similarly, another multiplex PCR assay was able to discriminate among the five B. suis biovars. A new TaqMan MGB real time PCR assays were also developed to detect Brucella with a sensitivity of 100 fg of DNA and a similar one step multiplex quantitative PCR assay can differentiate among B. abortus, B. melitensis, B. suis and B. ovis. The assessment of the genetic stability of the live attenuated vaccine B. melitensis Rev 1 was demonstrated by MLVA analysis (MLVA). However, MLVA can demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis in B. suis strains. The results of this work have originated five published papers, one international patent and two commercial kits.