The wide application of live attenuated vaccine strains is limited because of drawbacks of residual virulence, similar antigenenicity to virulent strain and the difficulty to differentiate vaccination and natural infection. In this study, we modified the vaccine strain to prevent the drawbacks. By using homologous recombination, we replaced the BP26 gene with the kanamycin gene in a live attenuated vaccine strain M5. The new tagged vaccine strain, M5DeltaBP26, was generated. The wild type strain and M5DeltaBP26 were used to infect macrophage and mice to compare their intracellular survival capability. According to the conservative sequence of dnaK and the deleted region of BP26, primers were designed to develop a duplex PCR for discriminating the wild type strain and M5DeltaBP26. A new tagged strain, M5DeltaBP26, was successfully constructed. The tagged strain could survive in both macrophage and mice, indicating the feasibility as live attenuated vaccine strain. Results from mice infection showed that, at 2 weeks p.i., 10(2.9) CFU of Brucella were isolated from M5 infected mice, whereas only 10(1.1) CFU of Brucella were isolated from M5DeltaBP26 infected mice (P < 0.01). At 3 weeks p. i., 10(2.2) CFU of Brucella whereas no M5DeltaBP26 were isolated. These results indicated that infection capability of M5DeltaBP26 was decreased. Based on the sequence differences between M5DeltaBP26 and M5, a new discriminating duplex PCR was developed. With the duplex PCR, only one product was amplified from M5DeltaBP26, by which it can be differentiated from wild type and virulent strains. The construction of tagged strain and the development of discriminating PCR provide a new candidate for further vaccine development.