IDENTIFICATION OF PATHOGENIC LEPTOSPIRA GENOSPECIES BY CONTINUOUS MONITORING OF FLUOROGENIC HYBRIDIZATION PROBES DURING RAPID-CYCLE PCR

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 p athogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptosp ira genus-specific amplification primers and pathogen-specific fluorog enic adjacent hybridization probes to be designed and synthesized, A P CR protocol was developed in which changes in fluorescence emission re sulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored, Nine strains of the pathogenic Leptospira genospecies could be differentiated from Lep tonema illini, Escherichia coli, and eight strains of Leptospira bifle xa. The PCR method was rapid, requiring 18 min for the completion of 4 5 cycles. It was also simple and flexible, as DNA templates prepared b y four different methods, including the simple boiling method, could b e used without adverse effects. Two hundred copies of target, equivale nt to 100 cells, could be detected.