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

Ferreira AS, Costa P, Rocha T, Amaro A, Vieira ML, Ahmed A, Thompson G, Hartskeerl RA, Inácio J - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

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Illustration of the real-time PCR amplification curves obtained during the testing of naturally-infected tissue samples.(A) Results of the β-actin and lipL32 targeted duplex amplification assay when testing representative samples from the wild rodents. The partial β-actin gene was amplified from all tissue samples (dark pink lines). Leptospiral DNA was detected in three samples by a positive amplification of the lipL32 gene (blue lines). A spiked positive control with L. interrogans (serovar Autumnalis, strain Akiyami) is shown (green line). (B) From the previous leptospiral positive amplification results, two samples were assessed as infected with L. borgpetersenii using the respective targeted amplification assay with probe TqM_bpn and flanking primers F_bpn and R_bpn1 (blue lines). The positive and negative controls are illustrated by the orange and red lines, respectively.
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pone-0112312-g002: Illustration of the real-time PCR amplification curves obtained during the testing of naturally-infected tissue samples.(A) Results of the β-actin and lipL32 targeted duplex amplification assay when testing representative samples from the wild rodents. The partial β-actin gene was amplified from all tissue samples (dark pink lines). Leptospiral DNA was detected in three samples by a positive amplification of the lipL32 gene (blue lines). A spiked positive control with L. interrogans (serovar Autumnalis, strain Akiyami) is shown (green line). (B) From the previous leptospiral positive amplification results, two samples were assessed as infected with L. borgpetersenii using the respective targeted amplification assay with probe TqM_bpn and flanking primers F_bpn and R_bpn1 (blue lines). The positive and negative controls are illustrated by the orange and red lines, respectively.

Mentions: DNA extracted from 27 kidney samples of wild rodents were analysed with the lipL32 and mammal β-actin targeted duplex assay (Table 2; Fig. 2A). Leptospiral DNA was detected in three samples, as demonstrated by a positive amplification of the lipL32 gene region (Table 2; Fig. 2A). Furthermore, the partial β-actin gene was amplified from all samples, showing that the PCR reactions were not significantly inhibited by potential contaminants. When tested with each of the L. interrogans, L. borgpetersenii, L. kirschneri and L. noguchii targeted probes/primers, only these three samples showed amplification (Table 2; Fig. 2B). Two of these DNA samples were identified as L. borgpetersenii and one sample as L. interrogans. Testing a pooled sample of kidney and liver tissues from a Patagonian mara, and a lung sample from an aborted swine fetus with the duplex PCR revealed a positive amplification for both samples (Table 2). Subsequent testing with the species-specific sets of probes and primers showed that the Patagonian mara was infected with L. interrogans and the swine fetus with L. kirschneri.


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

Ferreira AS, Costa P, Rocha T, Amaro A, Vieira ML, Ahmed A, Thompson G, Hartskeerl RA, Inácio J - PLoS ONE (2014)

Illustration of the real-time PCR amplification curves obtained during the testing of naturally-infected tissue samples.(A) Results of the β-actin and lipL32 targeted duplex amplification assay when testing representative samples from the wild rodents. The partial β-actin gene was amplified from all tissue samples (dark pink lines). Leptospiral DNA was detected in three samples by a positive amplification of the lipL32 gene (blue lines). A spiked positive control with L. interrogans (serovar Autumnalis, strain Akiyami) is shown (green line). (B) From the previous leptospiral positive amplification results, two samples were assessed as infected with L. borgpetersenii using the respective targeted amplification assay with probe TqM_bpn and flanking primers F_bpn and R_bpn1 (blue lines). The positive and negative controls are illustrated by the orange and red lines, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232388&req=5

pone-0112312-g002: Illustration of the real-time PCR amplification curves obtained during the testing of naturally-infected tissue samples.(A) Results of the β-actin and lipL32 targeted duplex amplification assay when testing representative samples from the wild rodents. The partial β-actin gene was amplified from all tissue samples (dark pink lines). Leptospiral DNA was detected in three samples by a positive amplification of the lipL32 gene (blue lines). A spiked positive control with L. interrogans (serovar Autumnalis, strain Akiyami) is shown (green line). (B) From the previous leptospiral positive amplification results, two samples were assessed as infected with L. borgpetersenii using the respective targeted amplification assay with probe TqM_bpn and flanking primers F_bpn and R_bpn1 (blue lines). The positive and negative controls are illustrated by the orange and red lines, respectively.
Mentions: DNA extracted from 27 kidney samples of wild rodents were analysed with the lipL32 and mammal β-actin targeted duplex assay (Table 2; Fig. 2A). Leptospiral DNA was detected in three samples, as demonstrated by a positive amplification of the lipL32 gene region (Table 2; Fig. 2A). Furthermore, the partial β-actin gene was amplified from all samples, showing that the PCR reactions were not significantly inhibited by potential contaminants. When tested with each of the L. interrogans, L. borgpetersenii, L. kirschneri and L. noguchii targeted probes/primers, only these three samples showed amplification (Table 2; Fig. 2B). Two of these DNA samples were identified as L. borgpetersenii and one sample as L. interrogans. Testing a pooled sample of kidney and liver tissues from a Patagonian mara, and a lung sample from an aborted swine fetus with the duplex PCR revealed a positive amplification for both samples (Table 2). Subsequent testing with the species-specific sets of probes and primers showed that the Patagonian mara was infected with L. interrogans and the swine fetus with L. kirschneri.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

Show MeSH
Related in: MedlinePlus