Direct Detection and Differentiation of Pathogenic <i>Leptospira</i> Species Using a Multi-Gene Targeted Real Time PCR Approach

<div><p>Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of <i>Leptospira</i>. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple <i>TaqMan</i>-based multi-gene targeted real-time PCR approach able to detect and differentiate <i>Leptospira interrogans</i>, <i>L. kirschneri</i>, <i>L. borgpeteresenii</i> and <i>L. noguchii</i>, which constitute the veterinary most relevant pathogenic species of <i>Leptospira</i>. The method uses sets of species-specific probes, and respective flanking primers, designed from <i>ompL1</i> and s<i>ecY</i> gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal <i>β-actin</i> and the leptospiral <i>lipL32</i> genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic <i>Leptospira</i> strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (<i>Mus</i> spp., <i>Rattus</i> spp., <i>Dolichotis patagonum</i> and <i>Sus domesticus</i>). Two samples were infected with <i>L. borgpetersenii</i>, two with <i>L. interrogans</i> and one with <i>L. kirschneri</i>. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis.</p>