Single tube identification and strain typing of Brucella melitensis by multiplex PCR

We report the development of a single-tube, multi-locus variable number tandem repeat analysis (MLVA) assay for simultaneous speciation and strain typing of Brucella, the etiologic agent of brucellosis. Our MLVA assay consists of eight loci, two of which are species-specific markers that allow for definitive identification of Brucella melitensis, B. abortus, and Brucella species while ruling out related pathogenic bacterial genera. The remaining six loci are moderately variable loci capable of discriminating between Brucella strains originating within our study area. We applied the assay to a collection of 110 B. melitensis isolates of primarily Mexican origin and to smaller sample sizes of four other Brucella species for a total of 161 isolates. Simpson's index of diversity was 0.985 for B. melitensis and 0.938 for B. abortus. The assay accurately distinguished seven epidemiologically-linked clusters of B. melitensis infections and ascertained the source of infection in several laboratory-acquired cases. This assay is accessible to limited-resource settings due to its technological and economical feasibility. The timely and accurate information provided by this assay will potentially aid brucellosis control efforts, improve patient outcomes, and reduce the occurrence of laboratory-acquired infections.