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A new tool for the detection and characterization of virulence plasmid was developed using the real-time PCR method. Two sets of primers, one targeting the major virulence plasmid (pYV) (YV-1, YV-2) and the other targeting a gene (YH-1, YH-2) located on the pYV (a 15-kb virulence plasmid) were designed to determine the presence and relative amount of pYV in various Y. enterocolitica strains. The designed primer sets were shown to be specific for pYV and not to cross-react with the other plasmids. The detection limit of the real-time PCR method was 10 copies per reaction, and the quantification range was from 10(3) to 10(9) copies. The method was successfully applied to detect pYV in 20 Y. enterocolitica strains. Three Y. enterocolitica strains harboring pYV (B/O:3, B/O:5, O:9) showed similar growth patterns in the real-time PCR assay. 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