IDENTIFICATION OF LEPTOSPIRA-INADAI BY CONTINUOUS MONITORING OF FLUORESCENCE DURING RAPID CYCLE PCR

Seven new Leptospira isolates from rats, a buffalo, and contaminated m edia showed either reactive serology against more than 1 serogroup or no reactive serology against a reference panel of 22 serovars in the m icroscopic agglutination test (MAT). Because of these inconclusive res ults, the 16S rDNA sequences of these isolates were determined and fou nd to resemble thar of the type strain of Leptospira inadai (L. inadai ), serovar lyme strain 10, which Is considered to be nonpathogenic for humans. Comparative analyses of other Leptospira 16S rDNA sequences f rom databases revealed a L. inadai-specific signature sequence, agains t which an amplification primer was designed. This primer when used in conjunction with an universal primer enabled the trial of a rapid PCR protocol in which fluorescence emissions due to binding of SYBR Green I dye to PCR products were continuously monitored during rapid therma l cycling. A melting curve acquired immediately after PCR was used to distinguish the intended product. The thermal cycling and continuous m onitoring of fluorescence emission were accomplished by the LightCycle r; the whole procedure of 30 PCR cycles and melting curve acquisition required only 20 minutes. The primer achieved the required specificity , as the intended PCR product resulted only from 6 confirmed L. inadai reference strains and 7 field isolates that had been verified as L. i nadai by the 16S rDNA sequencing, but not from 16 reference strains of Leptospira belonging to 7 other genospecies. Furthermore, these exper iments showed that the PCR protocol was robust because target DNA of d ifferent conditions, which were extracted by either 1 of the 4 methods used, could be detected.