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PLoS One. 2014 Nov 14;9(11):e112312. doi: 10.1371/journal.pone.0112312. eCollection 2014.

Direct detection and differentiation of pathogenic Leptospira species using a multi-gene targeted real time PCR approach.

Author information

1
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.
2
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.
3
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.
4
Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
5
WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, KIT Biomedical Research, Amsterdam, The Netherlands.
6
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.
7
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.

Abstract

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.

PMID:
25398140
PMCID:
PMC4232388
DOI:
10.1371/journal.pone.0112312
[Indexed for MEDLINE]
Free PMC Article

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