This test showed an analytical sensitivity of 10 homologous genome copies and when compared with culture proven leptospirosis Dapoxetine hydrochloride patients had a DSe and DSp of 100% and 93%, respectively. Here we describe the development and evaluation of a real-time PCR based on the SYBR Green Maprotiline hydrochloride technology targeting the secY gene. The primer pair we selected showed high specificity for detection of pathogenic leptospires, excluding all saprophytic strains tested and the vast majority of intermediate ones. The lack of amplification of DNA from most intermediate strains is not unexpected because their Leptospira species form a separate intermediate clade situated between pathogenic and saprophytic species. This signifies a difference in DNA composition compared to pathogenic species apparently resulting in a too low annealing capacity of the primers hampering the amplification. In our opinion, this is of little relevance for the diagnostic potential of the test. Infection of patients with intermediate leptospires is a relatively rare event and their pathogenic status is as yet doubtful. Even though some of the intermediate Leptospira spp. have been described as clinical isolates, virulence cannot convincingly be demonstrated. No cross-reaction was found with other micro-organisms, which is an important feature because secY is a house-keeping gene that has been demonstrated in many prokaryotic species. A major advantage of using the secY gene is its great phylogenetic potential. We recently demonstrated that a small 245 bp segment of secY, flanked by the primer pair G1/G2, had a high phylogenic power almost equaling that of the whole gene thus making it a feasible and interesting target for speciation by less sophisticated laboratories. We found that the 201 bp fragment of the gene amplified in this real-time PCR assay had a similar phylogenetic potential as the 245 bp G1/G2 restricted fragment, making this target an attractive alternative for sequencing and phylogeny following amplification in a conventional format. The real-time PCR was validated using the specific instructions from OIE.