In summary, the data presented here showed that the TaqMan probe T1 was suitable for use in the LightCycler-based 5' nuclease assay for identifying the pathogenic strains of L. interrogans. Sequence analyses of 23S rRNA genes of the pathogenic L. interrogans sensu lato and saprophytic L. biflexa enabled a pathogen-specific fluorogenic TaqMan probe to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from hybridization and hydrolysis of the probe were continuously monitored. Representative strains of the seven pathogenic Leptospira genospecies could be differentiated from six strains of L. biflexa and Lpn. illini. The PCR method was rapid, requiring 22 min for the completion of 45 cycles.