Direct detection and differentiation of pathogenic Leptospira species using a multi-gene targeted real time PCR approach.
Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. 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 TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 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 Leptospira 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 (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. 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.
Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal; Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal.
Ana Sofia
AS
Ferreira
Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal; Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
Pedro
P
Costa
Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal.
Teresa
T
Rocha
Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal.
Ana
A
Amaro
Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
Maria Luísa
ML
Vieira
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, KIT Biomedical Research, Amsterdam, The Netherlands.
Ahmed
A
Ahmed
Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal; Research Center in Biodiversity and Genetic Resources (CIBIO-ICETA), Universidade do Porto, Porto, Portugal.
Gertrude
G
Thompson
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, KIT Biomedical Research, Amsterdam, The Netherlands.
Rudy A
RA
Hartskeerl
Instituto Nacional de Investigação Agrária e Veterinária, I.P. (INIAV, I.P.), Unidade Estratégica de Investigação e Serviços em Produção e Saúde Animal, Lisboa, Portugal; School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, United Kingdom.
João
J
Inácio
D000818:Animals; D017931:DNA Primers / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D005798:Genes, Bacterial; D007919:Leptospira / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D015342:DNA Probes