Identifation methods of Brucella Melitensis

Abstract

Objective To establish genotyping methods for rapid identification of Brucella melitensis (B. melitensis) biovar 1, 2 and 3 and to verify these method. Methods Single nucleotide polymorphism of RpoB gene and tandem repeat sequence (TRS) Bru42 of standard reference strain 16M were used to design primers, then the RpoB-PCR and TRS-PCR method were established for identification of B. melitensis standard reference strains, these two methods were used to identify clinical isolates of B. melitensis and compared with the conventional methods. Results The results of B. melitensis standard reference strains (biotype 1, 2, 3) identified by RpoB-PCR and TRS-PCR were consistent with those of the conventional identification methods. Totally 50 clinical isolates [including B. melitensis biovar 1 (17), 2 (3) and 3 (30)] were identified as RpoB-2 genotype, only one B. melitensis biovar 1 strain was identified as RpoB-3 genotype. Genotype identification results of standard reference strains and clinical isolates with the same biotype were not exactly the same. Fothermore, TRS-PCR experiment displayed that 51 clinical isolates were all genotype 2 of B. melitensis(genotype TRS-2). Conclusions There is no clear relationship between biovars and genotypes within B. melitensis, and significant difference exists between B. melitensis standard reference strains and clinical isolates within RpoB gene. Bru42 can not be used for genotyping clinical isolates of B. melitensis. Key words: Brucella; Bacterial typing techniques; RpoB gene; Bru42 gene