DIRECT-DETECTION OF BRUCELLA SPP IN RAW-MILK BY PCR AND REVERSE HYBRIDIZATION WITH 16S-23S RIBOSOMAL-RNA SPACER PROBES

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, an d B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very hi gh (>99%) homology among the three species examined, Two genus-specifi c primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BR U-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity a nd sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), resp ectively, A method for direct detection of Brucella spp. in 1 mi of ra w milk was developed on the basis of enzymatic treatment of the milk c omponents and subsequent PCR and LiPA hybridization. After a single PC R, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained f or detection by agarose gel electrophoresis and LiPA, respectively, Ne sted PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.