REAL-TIME HOMOGENEOUS ASSAY OF RAPID CYCLE POLYMERASE-CHAIN-REACTION PRODUCT FOR IDENTIFICATION OF LEPTONEMA-ILLINI

Partial 16S rDNA sequences of eight Leptospira-like field isolates tha t reacted weakly or not at all to microscope agglutination test were f ound to be similar to the 16S rDNA sequence of the nonpathogen Leptone ma illini-type strain 3055. Comparison of these sequences with those o f 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 univ ersal reverse primer for developing a LightCycler-based rapid PCR prot ocol in which fluorescence emission due to the binding of SYBR green I dye to the amplified products was continuously monitored. A melting t emperature (T-m) determined from the melting curve of the amplified pr oduct immediately after PCR confirmed that the product was of leptonem a. The protocol for 24 samples consisting of 30 PCR cycles and melting curve acquisitions required 30 min to complete and agarose gel electr ophoresis of the PCR products was not necessary. The method was specif ic as PCR products were detected from the seven Leptonema reference st rains and the eight field isolates that had been previously verified a s Leptonema by 16S rDNA sequencing, but not from the two representativ e strains from each of the eight Leptospira genospecies tested. (C) 19 98 Academic Press.