Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals

Autores
Wehrendt, Diana Patricia; Gómez Bravo, Andrea; Cirignoli, Sebastián; Abril, Marcelo; Guhl, Felipe; Schijman, Alejandro Gabriel
Año de publicación
2017
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Chagas disease affects about 7 million people worldwide and is caused by the protozoan parasite Trypanosoma cruzi. In the argentine Chaco, an endemic area of Chagas disease, little is known about T. cruzi domestic and wild transmission cycles. Therefore, a field trial in rural areas near Añatuya, Santiago del Estero, for eco-epidemiological purposes, is under way. Due to the large number of samples we estimate to obtain in the trial, our goal was to develop a TaqMan multiplex PCR assay that simultaneously allowed T. cruzi detection and DNA integrity control. To achieve this, we choose the interphotoreceptor retinoid-binding protein (IRBP) gene, because it is highly conserved in all mammals and its use as a DNA integrity control in a conventional PCR was previously described. Based on an IRBP sequence alignment of several domestic and wild species, we designed a TaqMan probe to a highly conserved region within the amplified zone. The detection of satellite T. cruzi DNA was performed with the primers and probe already developed in our laboratory. Once the parameters for the multiplex PCR were established, the assay was tested in a total of 45 blood samples of wild mammals. So far, all samples analyzed were T. cruzi undetectable. Our DNA integrity control worked well for Conepatus chinga, Molossus sp, Lagostomus maximus, Lycalopex gymnocercus, Rattus rattus, Calomys sp, Galea musteloides and Leopardus geoffroyi. However, it did not work for Chaetophractus villosus, Tolypeutes matacus, Graomys chacoensis and Myotis sp. DNA integrity was tested by amplification of beta-actin in these cases. A sequence analysis to identify mutations that prevent amplification of the selected IRBP region in these species will be done. The performance of the multiplex PCR in domestic animal samples remains to be studied.
Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Gómez Bravo, Andrea. Fundación Mundo Sano; Argentina
Fil: Cirignoli, Sebastián. Fundación Mundo Sano; Argentina
Fil: Abril, Marcelo. Fundación Mundo Sano; Argentina
Fil: Guhl, Felipe. Centro de Investigaciones en Microbiología y Parasitología; Colombia
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; LXV Reunión Anual de la Sociedad Argentina de Inmunología; Reunión de la Sociedad Argentina de Andrología; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; XIX Reunión Anual de la Sociedad Argentina de Biología; XLIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; Reunión Anual de la Sociedad Argentina de Fisiología; Reunión de la Sociedad Argentina de Hematología y XXIX Reunión Anual de la Sociedad Argentina de Protozoología
Cuidad Autónoma de Buenos Aires
Argentina
Sociedad Argentina de Investigación Clínica
Sociedad Argentina de Investigación Bioquímica y Biología Molecular
Sociedad Argentina de Inmunología
Sociedad Argentina de Andrología
Sociedad Argentina de Biofísica
Sociedad Argentina de Biología
Sociedad Argentina de Farmacología Experimental
Sociedad Argentina de Fisiología
Sociedad Argentina de Hematología
Sociedad Argentina de Protozoología
Materia
CHAGAS DISEASE
TRYPANOSOMA CRUZI
TaqMan Multiplex PCR
WILD AND DOMESTIC RESERVOIRS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/275338
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/275338
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animalsWehrendt, Diana PatriciaGómez Bravo, AndreaCirignoli, SebastiánAbril, MarceloGuhl, FelipeSchijman, Alejandro GabrielCHAGAS DISEASETRYPANOSOMA CRUZITaqMan Multiplex PCRWILD AND DOMESTIC RESERVOIRShttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Chagas disease affects about 7 million people worldwide and is caused by the protozoan parasite Trypanosoma cruzi. In the argentine Chaco, an endemic area of Chagas disease, little is known about T. cruzi domestic and wild transmission cycles. Therefore, a field trial in rural areas near Añatuya, Santiago del Estero, for eco-epidemiological purposes, is under way. Due to the large number of samples we estimate to obtain in the trial, our goal was to develop a TaqMan multiplex PCR assay that simultaneously allowed T. cruzi detection and DNA integrity control. To achieve this, we choose the interphotoreceptor retinoid-binding protein (IRBP) gene, because it is highly conserved in all mammals and its use as a DNA integrity control in a conventional PCR was previously described. Based on an IRBP sequence alignment of several domestic and wild species, we designed a TaqMan probe to a highly conserved region within the amplified zone. The detection of satellite T. cruzi DNA was performed with the primers and probe already developed in our laboratory. Once the parameters for the multiplex PCR were established, the assay was tested in a total of 45 blood samples of wild mammals. So far, all samples analyzed were T. cruzi undetectable. Our DNA integrity control worked well for Conepatus chinga, Molossus sp, Lagostomus maximus, Lycalopex gymnocercus, Rattus rattus, Calomys sp, Galea musteloides and Leopardus geoffroyi. However, it did not work for Chaetophractus villosus, Tolypeutes matacus, Graomys chacoensis and Myotis sp. DNA integrity was tested by amplification of beta-actin in these cases. A sequence analysis to identify mutations that prevent amplification of the selected IRBP region in these species will be done. The performance of the multiplex PCR in domestic animal samples remains to be studied.Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gómez Bravo, Andrea. Fundación Mundo Sano; ArgentinaFil: Cirignoli, Sebastián. Fundación Mundo Sano; ArgentinaFil: Abril, Marcelo. Fundación Mundo Sano; ArgentinaFil: Guhl, Felipe. Centro de Investigaciones en Microbiología y Parasitología; ColombiaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaLXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; LXV Reunión Anual de la Sociedad Argentina de Inmunología; Reunión de la Sociedad Argentina de Andrología; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; XIX Reunión Anual de la Sociedad Argentina de Biología; XLIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; Reunión Anual de la Sociedad Argentina de Fisiología; Reunión de la Sociedad Argentina de Hematología y XXIX Reunión Anual de la Sociedad Argentina de ProtozoologíaCuidad Autónoma de Buenos AiresArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de Investigación Bioquímica y Biología MolecularSociedad Argentina de InmunologíaSociedad Argentina de AndrologíaSociedad Argentina de BiofísicaSociedad Argentina de BiologíaSociedad Argentina de Farmacología ExperimentalSociedad Argentina de FisiologíaSociedad Argentina de HematologíaSociedad Argentina de ProtozoologíaFundación Revista Medicina2017info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/vnd.openxmlformats-officedocument.wordprocessingml.documentapplication/pdfhttp://hdl.handle.net/11336/275338Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals; LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; LXV Reunión Anual de la Sociedad Argentina de Inmunología; Reunión de la Sociedad Argentina de Andrología; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; XIX Reunión Anual de la Sociedad Argentina de Biología; XLIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; Reunión Anual de la Sociedad Argentina de Fisiología; Reunión de la Sociedad Argentina de Hematología y XXIX Reunión Anual de la Sociedad Argentina de Protozoología; Cuidad Autónoma de Buenos Aires; Argentina; 2017; 190-1901669-9106CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.medicinabuenosaires.com/indices-de-2010-a-2019/Internacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-12-17T14:55:44Zoai:ri.conicet.gov.ar:11336/275338instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-12-17 14:55:44.655CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
title Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
spellingShingle Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
Wehrendt, Diana Patricia
CHAGAS DISEASE
TRYPANOSOMA CRUZI
TaqMan Multiplex PCR
WILD AND DOMESTIC RESERVOIRS
title_short Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
title_full Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
title_fullStr Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
title_full_unstemmed Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
title_sort Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals
dc.creator.none.fl_str_mv Wehrendt, Diana Patricia
Gómez Bravo, Andrea
Cirignoli, Sebastián
Abril, Marcelo
Guhl, Felipe
Schijman, Alejandro Gabriel
author Wehrendt, Diana Patricia
author_facet Wehrendt, Diana Patricia
Gómez Bravo, Andrea
Cirignoli, Sebastián
Abril, Marcelo
Guhl, Felipe
Schijman, Alejandro Gabriel
author_role author
author2 Gómez Bravo, Andrea
Cirignoli, Sebastián
Abril, Marcelo
Guhl, Felipe
Schijman, Alejandro Gabriel
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv CHAGAS DISEASE
TRYPANOSOMA CRUZI
TaqMan Multiplex PCR
WILD AND DOMESTIC RESERVOIRS
topic CHAGAS DISEASE
TRYPANOSOMA CRUZI
TaqMan Multiplex PCR
WILD AND DOMESTIC RESERVOIRS
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Chagas disease affects about 7 million people worldwide and is caused by the protozoan parasite Trypanosoma cruzi. In the argentine Chaco, an endemic area of Chagas disease, little is known about T. cruzi domestic and wild transmission cycles. Therefore, a field trial in rural areas near Añatuya, Santiago del Estero, for eco-epidemiological purposes, is under way. Due to the large number of samples we estimate to obtain in the trial, our goal was to develop a TaqMan multiplex PCR assay that simultaneously allowed T. cruzi detection and DNA integrity control. To achieve this, we choose the interphotoreceptor retinoid-binding protein (IRBP) gene, because it is highly conserved in all mammals and its use as a DNA integrity control in a conventional PCR was previously described. Based on an IRBP sequence alignment of several domestic and wild species, we designed a TaqMan probe to a highly conserved region within the amplified zone. The detection of satellite T. cruzi DNA was performed with the primers and probe already developed in our laboratory. Once the parameters for the multiplex PCR were established, the assay was tested in a total of 45 blood samples of wild mammals. So far, all samples analyzed were T. cruzi undetectable. Our DNA integrity control worked well for Conepatus chinga, Molossus sp, Lagostomus maximus, Lycalopex gymnocercus, Rattus rattus, Calomys sp, Galea musteloides and Leopardus geoffroyi. However, it did not work for Chaetophractus villosus, Tolypeutes matacus, Graomys chacoensis and Myotis sp. DNA integrity was tested by amplification of beta-actin in these cases. A sequence analysis to identify mutations that prevent amplification of the selected IRBP region in these species will be done. The performance of the multiplex PCR in domestic animal samples remains to be studied.
Fil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Gómez Bravo, Andrea. Fundación Mundo Sano; Argentina
Fil: Cirignoli, Sebastián. Fundación Mundo Sano; Argentina
Fil: Abril, Marcelo. Fundación Mundo Sano; Argentina
Fil: Guhl, Felipe. Centro de Investigaciones en Microbiología y Parasitología; Colombia
Fil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; LXV Reunión Anual de la Sociedad Argentina de Inmunología; Reunión de la Sociedad Argentina de Andrología; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; XIX Reunión Anual de la Sociedad Argentina de Biología; XLIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; Reunión Anual de la Sociedad Argentina de Fisiología; Reunión de la Sociedad Argentina de Hematología y XXIX Reunión Anual de la Sociedad Argentina de Protozoología
Cuidad Autónoma de Buenos Aires
Argentina
Sociedad Argentina de Investigación Clínica
Sociedad Argentina de Investigación Bioquímica y Biología Molecular
Sociedad Argentina de Inmunología
Sociedad Argentina de Andrología
Sociedad Argentina de Biofísica
Sociedad Argentina de Biología
Sociedad Argentina de Farmacología Experimental
Sociedad Argentina de Fisiología
Sociedad Argentina de Hematología
Sociedad Argentina de Protozoología
description Chagas disease affects about 7 million people worldwide and is caused by the protozoan parasite Trypanosoma cruzi. In the argentine Chaco, an endemic area of Chagas disease, little is known about T. cruzi domestic and wild transmission cycles. Therefore, a field trial in rural areas near Añatuya, Santiago del Estero, for eco-epidemiological purposes, is under way. Due to the large number of samples we estimate to obtain in the trial, our goal was to develop a TaqMan multiplex PCR assay that simultaneously allowed T. cruzi detection and DNA integrity control. To achieve this, we choose the interphotoreceptor retinoid-binding protein (IRBP) gene, because it is highly conserved in all mammals and its use as a DNA integrity control in a conventional PCR was previously described. Based on an IRBP sequence alignment of several domestic and wild species, we designed a TaqMan probe to a highly conserved region within the amplified zone. The detection of satellite T. cruzi DNA was performed with the primers and probe already developed in our laboratory. Once the parameters for the multiplex PCR were established, the assay was tested in a total of 45 blood samples of wild mammals. So far, all samples analyzed were T. cruzi undetectable. Our DNA integrity control worked well for Conepatus chinga, Molossus sp, Lagostomus maximus, Lycalopex gymnocercus, Rattus rattus, Calomys sp, Galea musteloides and Leopardus geoffroyi. However, it did not work for Chaetophractus villosus, Tolypeutes matacus, Graomys chacoensis and Myotis sp. DNA integrity was tested by amplification of beta-actin in these cases. A sequence analysis to identify mutations that prevent amplification of the selected IRBP region in these species will be done. The performance of the multiplex PCR in domestic animal samples remains to be studied.
publishDate 2017
dc.date.none.fl_str_mv 2017
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Reunión
Journal
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/275338
Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals; LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; LXV Reunión Anual de la Sociedad Argentina de Inmunología; Reunión de la Sociedad Argentina de Andrología; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; XIX Reunión Anual de la Sociedad Argentina de Biología; XLIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; Reunión Anual de la Sociedad Argentina de Fisiología; Reunión de la Sociedad Argentina de Hematología y XXIX Reunión Anual de la Sociedad Argentina de Protozoología; Cuidad Autónoma de Buenos Aires; Argentina; 2017; 190-190
1669-9106
CONICET Digital
CONICET
url http://hdl.handle.net/11336/275338
identifier_str_mv Development of a TaqMan Multiplex Real Time PCR assay for the detection of Trypanosoma cruzi in blood samples of wild and domestic animals; LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; LXV Reunión Anual de la Sociedad Argentina de Inmunología; Reunión de la Sociedad Argentina de Andrología; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; XIX Reunión Anual de la Sociedad Argentina de Biología; XLIX Reunión Anual de la Sociedad Argentina de Farmacología Experimental; Reunión Anual de la Sociedad Argentina de Fisiología; Reunión de la Sociedad Argentina de Hematología y XXIX Reunión Anual de la Sociedad Argentina de Protozoología; Cuidad Autónoma de Buenos Aires; Argentina; 2017; 190-190
1669-9106
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.medicinabuenosaires.com/indices-de-2010-a-2019/
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eu_rights_str_mv openAccess
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dc.format.none.fl_str_mv application/pdf
application/vnd.openxmlformats-officedocument.wordprocessingml.document
application/pdf
dc.coverage.none.fl_str_mv Internacional
dc.publisher.none.fl_str_mv Fundación Revista Medicina
publisher.none.fl_str_mv Fundación Revista Medicina
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
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repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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