COMPARISON OF 2 PCR METHODS FOR RAPID IDENTIFICATION OF LEPTOSPIRA GENOSPECIES INTERROGANS

Based on (i) an analysis of Leptospira 16S rDNA sequences determined b y us and of those from databases and (ii) a previously published findi ng that restriction fragment length polymorphisms (RFLPs) within the L eptospira 16S and 23S rDNA were detected by nine restriction enzymes a nd these RFLPs allowed categorisation of Leptospira into eight genospe cies, we predicted that one particular DdeI restriction site polymorph ism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans. Two PCR- based methods, namely allele-specific amplification (ASA) and PCR-RFLP , were tested for the rapid detection of the DdeI restriction site pol ymorphism. One or two representative strains from each of nine genospe cies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were rested by PCR-RFLP. Our experiments showed tha t the ASA method was not as specific as intended, but the PCR-RFLP met hod was useful for rapid identifications of the genospecies interrogan s. We have not only confirmed a previous finding and extended the numb er of samples particularly from the genospecies biflexa, weilii, and i nadai, but also simplified a previous PCR-RFLP protocol.