Two new primer sets of a 766-and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates Brucella canis and Brucella microti from Brucella suis strains and also may differentiate all of the 10 Brucella species.The alphaproteobacterial genus Brucella consists of 10 species: B. abortus, B. canis, B. suis, B. ovis, B. neotomae, B. melitensis, B. ceti, B. pinnipedialis, B. microti, and B. inopinata (3, 5, 16, 17). Brucella species show a host preference, but some strains can be transmitted among a variety of animals, including humans (11,12,14,18).To accelerate effective prevention and control of brucellosis, a fast and accurate identification method is necessary. Many studies have developed PCR-based assays for the discrimination of Brucella species (2,4,7,8,10). Recently, López-Goñi et al. reported that the Bruce-ladder PCR assay could differentiate most Brucella species, including marine mammal and vaccine strains B. abortus S19, B. abortus RB51, and B. melitensis Rev.1 (6, 10, 13). However, these assays did not solve the problem of erroneous identification of B. canis isolates (such as B. suis) and basic differentiation between two marine mammal Brucella species (B. ceti and B. pinnipedialis) (9, 13, 15). Therefore, the aim of this study was to develop a fast, simple, and accurate one-step multiplex PCR assay to differentiate 10 Brucella species using 22 reference strains and 106 field isolates in South Korea.To discriminate between B. canis and B. suis, alignments of their whole-genome sequence or each biovar short-gun

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BackgroundA Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to B. abortus isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates.ResultsA total of 177 isolates originating from 105 cattle farms for the period 1996 to 2008 were selected as representatives for the nine provinces of South Korea. A dendrogram of strain relatedness was constructed in accordance with the number of tandem repeat units for 17 loci so that it was possible to trace back in the restricted areas. Even in a farm contaminated by one source, however, the Brucella isolates showed an increase or decrease in one TRs copy number at some loci with high DI values. Moreover, those 17 loci was confirmed in stability via in-vitro and in-vivo passage, and found to be sufficiently stable markers that can readily identify the inoculated strain even if minor changes were detected. In the parsimony analysis with foreign Brucella isolates, domestic isolates were clustered distinctively, and located near the Central and Southern American isolates.ConclusionThe MLVA assay has enough discrimination power in the Brucella species level and can be utilized as a tool for the epidemiological trace-back of the B. abortus isolates. But it is important to consider that Brucella isolates may be capable of undergoing minor changes at some loci in the course of infection or in accordance with the changes of the host.

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