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Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes

Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-P...

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Autores principales: Xu, Chuanling, Loftis, Amanda, Ahluwalia, Sudhir K., Gao, Dongya, Verma, Ashutosh, Wang, Chengming, Kaltenboeck, Bernhard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933566/
https://www.ncbi.nlm.nih.gov/pubmed/24586833
http://dx.doi.org/10.1371/journal.pone.0089507
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author Xu, Chuanling
Loftis, Amanda
Ahluwalia, Sudhir K.
Gao, Dongya
Verma, Ashutosh
Wang, Chengming
Kaltenboeck, Bernhard
author_facet Xu, Chuanling
Loftis, Amanda
Ahluwalia, Sudhir K.
Gao, Dongya
Verma, Ashutosh
Wang, Chengming
Kaltenboeck, Bernhard
author_sort Xu, Chuanling
collection PubMed
description Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis.
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spelling pubmed-39335662014-02-25 Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes Xu, Chuanling Loftis, Amanda Ahluwalia, Sudhir K. Gao, Dongya Verma, Ashutosh Wang, Chengming Kaltenboeck, Bernhard PLoS One Research Article Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis. Public Library of Science 2014-02-24 /pmc/articles/PMC3933566/ /pubmed/24586833 http://dx.doi.org/10.1371/journal.pone.0089507 Text en © 2014 Xu et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Xu, Chuanling
Loftis, Amanda
Ahluwalia, Sudhir K.
Gao, Dongya
Verma, Ashutosh
Wang, Chengming
Kaltenboeck, Bernhard
Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes
title Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes
title_full Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes
title_fullStr Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes
title_full_unstemmed Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes
title_short Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes
title_sort diagnosis of canine leptospirosis by a highly sensitive fret-pcr targeting the lig genes
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933566/
https://www.ncbi.nlm.nih.gov/pubmed/24586833
http://dx.doi.org/10.1371/journal.pone.0089507
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