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). We designed 8 pairs of oligonucleotide primers for a multiplex PCR assay (see Table 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue4/) carried out with a 25-μL reaction mixture containing 1 μL of template DNA (from standard methods or heat lysis of cultures), 400 μM each deoxynucleoside triphosphate (Promega Corp.), 3 mM MgCl2, 1.5 U of Immolase DNA polymerase and its amplification buffer (Bioline Ltd), and 6.25 pmol of each primer. Thermal cycling was accomplished with a GeneAmp® PCR System 2700 (Applied Biosystems). After initial denaturation at 95 °C for 7 min, the PCR profile was as follows: 35 s of template denaturation at 95 °C, 45 s of primer annealing at 64 °C, and 180 s of primer extension at 72 °C, for a total of 25 cycles, with a final extension at 72 °C for 6 min. We analyzed PCR products by calibrated 1.5% agarose electrophoresis. A representative example of the multiplex PCR results is presented in Fig. 11 . PCR with B. melitensis DNA amplified 6 fragments: 1682, 1071, 794, 587, 450, and 152 bp. A specific additional 1320-bp fragment was amplified, and the 450-bp fragment was absent in Brucella strains isolated from marine mammals. B. ovis was distinguished by the absence of the 1682-bp fragment, B. abortus by the absence of the 1071-bp fragment, B. suis by the presence of an additional 272-bp fragment (also present in B. canis and B. neotomae), B. canis by the absence of the 794-bp fragment, and B. neotomae by the absence of the 152-bp fragment. The vaccine strain B. abortus RB51 was readily distinguished by a specific additional 2524-bp fragment and by the absence of the 1682-bp fragment, and the vaccine strain B. abortus S19 did not produce the 587-bp fragment common to all other Brucella strains tested. B. melitensis Rev1 was distinguished by amplification of a specific 218-bp fragment only with DNA from this strain. We evaluated the test with 72 Brucella reference strains and isolates from humans and animals (see Table 2 in the online Data Supplement). All strains and biovars from the same species gave the same profile. No amplification products were obtained with DNAs from 23 bacteria phylogenetically or serologically related to Brucella (see Table 2 in the online Data Supplement), demonstrating that this test was highly specific for Brucella. A major advantage of our assay is that it can identify and differentiate all known Brucella species, including the Brucella isolates from marine mammals and the vaccine strains S19, RB51, and Rev1. This robust assay for the identification of Brucella is fast and safe and requires minimal sample preparation. It can be used in reference centers and in basic microbiology laboratories worldwide. Identification and differentiation of all Brucella species and vaccine strains by multiplex PCR. Lane 1, Brucella isolated from marine mammals (IS711 insertion in BMEI0535-BMEI0536); lane 2, B. melitensis; lane 3, vaccine strain B. melitensis Rev1 (point mutation in BMEI0752); lane 4, B. ovis (deletion of 15 079 bp in BMEI0993-BMEI1012); lane 5, B. abortus (deletion of 25 061 bp in BMEII826-BMEII0850); lane 6, vaccine strain B. abortus RB51 (IS711 insertion in BMEI0998); lane 7, vaccine strain B. abortus S19 (deletion of 702 bp in BMEII0427-BMEII0428); lane 8, B. suis (deletion of 2653 bp in BR0951-BR0955 in B. melitensis and B. abortus); lane 9, B. canis (deletion of 976 bp in BME1435); lane 10, B. neotomae (deletion of 2203 bp in BMEII0986-BMEII0988); lane 11, 1 kb plus DNA ladder (Invitrogen Ltd.), used as molecular size markers.