Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts

A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can f...

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Detalles Bibliográficos
Autor: Ramsey, Janine
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2019
País:México
Institución:Instituto Nacional de Salud Pública
Repositorio:Repositorio Institucional Abierto de Conocimiento en Salud Pública
Idioma:español
OAI Identifier:oai:repositorio.insp.mx:20.500.12096/7661
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884757/
https://www.doi.org/10.1186/s13071-019-3817-9.
http://repositorio.insp.mx:8080/jspui/handle/20.500.12096/7661
Access Level:acceso abierto
Palabra clave:AnimalsAnimals, Domestic parasitologyAnimals, Wild parasitologyChagas Disease diagnosisChagas Disease veterinary,DNA Primers geneticsDNA Probes geneticsDNA, Protozoan isolation purificationDNA, Satellite isolation purificationDisease Reservoirs parasitology,Eye Proteins geneticsMammals parasitologyReal-Time Polymerase Chain Reaction methodsRetinol-Binding Proteins geneticsSensitivity and SpecificityTrypanosoma cruzi,SD
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spelling Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hostsRamsey, JanineAnimalsAnimals, Domestic parasitologyAnimals, Wild parasitologyChagas Disease diagnosisChagas Disease veterinary,DNA Primers geneticsDNA Probes geneticsDNA, Protozoan isolation purificationDNA, Satellite isolation purificationDisease Reservoirs parasitology,Eye Proteins geneticsMammals parasitologyReal-Time Polymerase Chain Reaction methodsRetinol-Binding Proteins geneticsSensitivity and SpecificityTrypanosoma cruzi,SDinfo:eu-repo/classification/cti/3A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. Results: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. Conclusions: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.ESPM INSP2022-02-16T04:20:21Z2022-02-16T04:20:21Z2019info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionpdfsicabi.insp.mx:2019-Nonehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884757/https://www.doi.org/10.1186/s13071-019-3817-9.http://repositorio.insp.mx:8080/jspui/handle/20.500.12096/7661reponame:Repositorio Institucional Abierto de Conocimiento en Salud Públicainstname:Instituto Nacional de Salud Públicainstacron:INSPspanacionalinfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-nd/4.0oai:repositorio.insp.mx:20.500.12096/76612024-08-27T22:04:39Z
dc.title.none.fl_str_mv Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
title Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
spellingShingle Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
Ramsey, Janine
AnimalsAnimals, Domestic parasitologyAnimals, Wild parasitologyChagas Disease diagnosisChagas Disease veterinary,DNA Primers geneticsDNA Probes geneticsDNA, Protozoan isolation purificationDNA, Satellite isolation purificationDisease Reservoirs parasitology,Eye Proteins geneticsMammals parasitologyReal-Time Polymerase Chain Reaction methodsRetinol-Binding Proteins geneticsSensitivity and SpecificityTrypanosoma cruzi,SD
info:eu-repo/classification/cti/3
title_short Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
title_full Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
title_fullStr Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
title_full_unstemmed Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
title_sort Development and evaluation of a duplex TaqMan qPCR assay for detection and quantifcation of Trypanosoma cruzi infection in domestic and sylvatic reservoir hosts
dc.creator.none.fl_str_mv Ramsey, Janine
author Ramsey, Janine
author_facet Ramsey, Janine
author_role author
dc.subject.none.fl_str_mv AnimalsAnimals, Domestic parasitologyAnimals, Wild parasitologyChagas Disease diagnosisChagas Disease veterinary,DNA Primers geneticsDNA Probes geneticsDNA, Protozoan isolation purificationDNA, Satellite isolation purificationDisease Reservoirs parasitology,Eye Proteins geneticsMammals parasitologyReal-Time Polymerase Chain Reaction methodsRetinol-Binding Proteins geneticsSensitivity and SpecificityTrypanosoma cruzi,SD
info:eu-repo/classification/cti/3
topic AnimalsAnimals, Domestic parasitologyAnimals, Wild parasitologyChagas Disease diagnosisChagas Disease veterinary,DNA Primers geneticsDNA Probes geneticsDNA, Protozoan isolation purificationDNA, Satellite isolation purificationDisease Reservoirs parasitology,Eye Proteins geneticsMammals parasitologyReal-Time Polymerase Chain Reaction methodsRetinol-Binding Proteins geneticsSensitivity and SpecificityTrypanosoma cruzi,SD
info:eu-repo/classification/cti/3
description A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. Results: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. Conclusions: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.
publishDate 2019
dc.date.none.fl_str_mv 2019
2022-02-16T04:20:21Z
2022-02-16T04:20:21Z
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv sicabi.insp.mx:2019-None
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884757/
https://www.doi.org/10.1186/s13071-019-3817-9.
http://repositorio.insp.mx:8080/jspui/handle/20.500.12096/7661
identifier_str_mv sicabi.insp.mx:2019-None
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884757/
https://www.doi.org/10.1186/s13071-019-3817-9.
http://repositorio.insp.mx:8080/jspui/handle/20.500.12096/7661
dc.language.none.fl_str_mv spa
language spa
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-nd/4.0
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0
dc.format.none.fl_str_mv pdf
dc.coverage.none.fl_str_mv nacional
dc.publisher.none.fl_str_mv ESPM INSP
publisher.none.fl_str_mv ESPM INSP
dc.source.none.fl_str_mv reponame:Repositorio Institucional Abierto de Conocimiento en Salud Pública
instname:Instituto Nacional de Salud Pública
instacron:INSP
instname_str Instituto Nacional de Salud Pública
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