Abstract: Three pairs of primers were designed according to the specific gene BCSP31 of Brucella genus and insert sequence IS711. The DNA of B.abortus 544A, B.abortus104M and B.melitensis 16M strains were used as the positive control to establish the multiplex PCR assay. Developed a multiplex PCR procedure to identify two major species of the genus Brucella simultaneously. Bands of 301, 114 bp could be amplified from strains of B.abortus, and 301，253 bp from B.melitensis. The multiplex PCR assay could detect as low as 100 pg of mixed B.abortus 544A and B.melitensis 16M DNA. No specific bands could be amplified from control bacteria such as E.coli O157：H7 and Yersinia enterocolitica. 106 cow feces samples were determined by the developed multiplex PCR assay, and total 9 samples displied the B.abortus positive. Rose bengal plate agglutination test was used as controlled trial, and 9 samples were Brucella positive. The results indicated that the established multiplex PCR assay was highly sensitive and specific.This assay provided a molecular detection tool to differential diagnosis for brucellosis.

Key words: B.abortus; B.melitensis; BCSP31 gene; IS711 gene; multiplex PCR