Partial 16S rDNA sequences of eight Leptospira-like field isolates that reacted weakly or not at all to microscope agglutination test were found to be similar to the 16S rDNA sequence of the nonpathogen Leptonema illini-type strain 3055. Comparison of these sequences with those of Leptospira 16S rDNA sequences revealed a Leptonema species signature sequence for which a forward amplification primer was designed. This primer was used in conjunction with a bacterial-specific 16S rDNA universal reverse primer for developing a LightCycler-based rapid PCR protocol in which fluorescence emission due to the binding of SYBR green I dye to the amplified products was continuously monitored. A melting temperature (T(m)) determined from the melting curve of the amplified product immediately after PCR confirmed that the product was of Leptonema. The protocol for 24 samples consisting of 30 PCR cycles and melting curve acquisitions required 30 min to complete and agarose gel electrophoresis of the PCR products was not necessary. The method was specific as PCR products were detected from the seven Leptonema reference strains and the eight field isolates that had been previously verified as Leptonema by 16S rDNA sequencing, but not from the two representative strains from each of the eight Leptospira genospecies tested.