Multiplex PCR Assay for the Identification and Differentiation of all Brucella Species and the Vaccine Strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1

The genus Brucella consists of 6 recognized bacterial species (1) and 2 proposed new species recently isolated from marine mammals (2). Some species have several biovars or biotypes, distinguishable by time-consuming analysis of phenotypic characteristics (3). Such analyses are subject to variable interpretations and must be performed by skilled technicians who are at high risk because most clinical Brucella strains are highly pathogenic. Accurate typing procedures are critical for eradication and control of disease-causing organisms, but DNA-based techniques are challenging because the various Brucella species and strains have a high degree of genetic homology, as demonstrated by whole-genome comparative analyses of the 3 Brucella genomes sequenced, Brucella melitensis (4), Brucella suis (5), and Brucella abortus (6). Unique fragments occur among the different Brucella genomes, however, which allowed us to develop a rapid, specific single-test-tube assay for molecular identification of all known Brucella species. We used the following species- or strain-specific genetic differences to design PCR primers: (a) a 25-kb DNA deletion leading to the loss of omp31 gene in the reference strains of all B. abortus biovars (6)(7); (b) a 15-kb deletion comprising omp25b and wboA-wboB genes in the Brucella ovis species (6)(7); (c) a wboA gene disruption by an IS711 element in the B. abortus vaccine strain RB51 (8); (d) a 702-bp deletion in the ery operon in the vaccine strain B. abortus S19 (9); (e) a specific mutation in the rpsL gene of the vaccine strain B. melitensis Rev1 that differentiates it from the B. melitensis reference strain (10); (f) a 976-bp deletion in chromosome I specific to Brucella canis (7); (g) a 2.2-kb deletion in chromosome II specific to Brucella neotomae (7); (h) a 2.6-kb fragment in B. suis, but not in B. abortus or B. melitensis (6)(7); and (i) an IS711 element downstream of the bp26 gene in Brucella spp. isolated from marine mammals (11).