Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs
- Autores
- Wehrendt, Diana Patricia; Gómez Bravo, Andrea; Pech May, Angélica del Rosario; Ramsey, Janine; Cura, Carolina Inés; Curto, Maria de Los Angeles; Abril, M.; Guhl, F.; Schijman, Alejandro Gabriel
- Año de publicación
- 2018
- Idioma
- inglés
- Tipo de recurso
- documento de conferencia
- Estado
- versión publicada
- Descripción
- A major question of epidemiological relevance in Chagas disease studies is to understand the dynamics ofT.cruzi infection in sylvatic and domestic transmission cycles and to trace the origins of (re)emerging casesin areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity and molecularapproaches, such as the polymerase chain reaction may ll in this gap, provided that a standardizedmethod can be developed and validated.We have developed two duplex Real Time PCR assays for sensitive detection of T.cruzi satellite or minicirclerepetitive sequences in blood samples from mammal reservoirs, incorporating an internal amplicationstandard that allows distinction of false negative PCR ndings due to inadequate conditions of storageand transport of samples, DNA degradation during nucleic acid purication and/or inhibition of PCR byinterfering substances present in the sample. The housekeeping gene that encodes the interphotoreceptorretinoid-binding protein (IRBP) has been selected as internal standard, because it is highly conserved amongall mammal species and its usefulness as a DNA integrity control was previously reported in a conventionalPCR protocol. Based on the alignment of the IRBP sequence available for several domestic and wild reservoirspecies, we designed primers and a TaqMan probe to a highly conserved region. The analytical sensitivitywas 0.01 par. eq/mL and 0.1 par.eq/mL for satDNA/IRBP duplex and kDNA/IRBP duplex, respectively, astested with two series of canine blood spiked with known concentrations of Silvio X10 (Tc I) and CL-Brener(Tc VI) cultured epimastigotes. The assays were evaluated in DNA extracts from blood samples of 87 wildand 147 domestic animals. Our DNA integrity control worked well for wild reservoir species including smallrodents (Akodon toba, Galea leucoblephara, Rattus rattus), opossums (Didelphis virginiana, D. marsupialis),bats (Tadarida brasiliensis, Promops nasutus, Desmodus rotundus) and other mammals such as the skunk,viscacha, wildcat, brown brocket deer, hare and Pampas fox. Mean Ct values for IRBP amplication variedamong species between 24 and 33. For domestic reservoirs, IRBP amplied in dog, cat, cow, sheep, goat,horse and mule specimens with mean Ct values between 23 and 25. In T.cruzi infected cases the assaysallowed quantication of parasitic loads.Our results promote the use of these methods for rapid and accurate screening of T.cruzi infection in dierentspecies of reservoirs.
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: Pech May, Angélica del Rosario. Instituto Nacional de Salud Pública; México. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Ramsey, Janine. Instituto Nacional de Salud Pública; México
Fil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Abril, M.. Fundación Mundo Sano; Argentina
Fil: Guhl, F.. Universidad de los Andes; 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
XXX Reunión Anual de la Sociedad Argentina de Protozoología
Resistencia
Argentina
Sociedad Argentina de Protozoología - Materia
-
TAQMAN
Diagnostics
Trypanosoma cruzi
Reservoirs - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
.jpg)
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/201729
Ver los metadatos del registro completo
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Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirsWehrendt, Diana PatriciaGómez Bravo, AndreaPech May, Angélica del RosarioRamsey, JanineCura, Carolina InésCurto, Maria de Los AngelesAbril, M.Guhl, F.Schijman, Alejandro GabrielTAQMANDiagnosticsTrypanosoma cruziReservoirshttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1A major question of epidemiological relevance in Chagas disease studies is to understand the dynamics ofT.cruzi infection in sylvatic and domestic transmission cycles and to trace the origins of (re)emerging casesin areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity and molecularapproaches, such as the polymerase chain reaction may ll in this gap, provided that a standardizedmethod can be developed and validated.We have developed two duplex Real Time PCR assays for sensitive detection of T.cruzi satellite or minicirclerepetitive sequences in blood samples from mammal reservoirs, incorporating an internal amplicationstandard that allows distinction of false negative PCR ndings due to inadequate conditions of storageand transport of samples, DNA degradation during nucleic acid purication and/or inhibition of PCR byinterfering substances present in the sample. The housekeeping gene that encodes the interphotoreceptorretinoid-binding protein (IRBP) has been selected as internal standard, because it is highly conserved amongall mammal species and its usefulness as a DNA integrity control was previously reported in a conventionalPCR protocol. Based on the alignment of the IRBP sequence available for several domestic and wild reservoirspecies, we designed primers and a TaqMan probe to a highly conserved region. The analytical sensitivitywas 0.01 par. eq/mL and 0.1 par.eq/mL for satDNA/IRBP duplex and kDNA/IRBP duplex, respectively, astested with two series of canine blood spiked with known concentrations of Silvio X10 (Tc I) and CL-Brener(Tc VI) cultured epimastigotes. The assays were evaluated in DNA extracts from blood samples of 87 wildand 147 domestic animals. Our DNA integrity control worked well for wild reservoir species including smallrodents (Akodon toba, Galea leucoblephara, Rattus rattus), opossums (Didelphis virginiana, D. marsupialis),bats (Tadarida brasiliensis, Promops nasutus, Desmodus rotundus) and other mammals such as the skunk,viscacha, wildcat, brown brocket deer, hare and Pampas fox. Mean Ct values for IRBP amplication variedamong species between 24 and 33. For domestic reservoirs, IRBP amplied in dog, cat, cow, sheep, goat,horse and mule specimens with mean Ct values between 23 and 25. In T.cruzi infected cases the assaysallowed quantication of parasitic loads.Our results promote the use of these methods for rapid and accurate screening of T.cruzi infection in dierentspecies of reservoirs.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: Pech May, Angélica del Rosario. Instituto Nacional de Salud Pública; México. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ramsey, Janine. Instituto Nacional de Salud Pública; MéxicoFil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Abril, M.. Fundación Mundo Sano; ArgentinaFil: Guhl, F.. Universidad de los Andes; ColombiaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXX Reunión Anual de la Sociedad Argentina de ProtozoologíaResistenciaArgentinaSociedad Argentina de ProtozoologíaSociedad Argentina de ProtozoologíaLucero, Raul Horacio2018info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónBookhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/vnd.openxmlformats-officedocument.wordprocessingml.documentapplication/pdfhttp://hdl.handle.net/11336/201729Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs; XXX Reunión Anual de la Sociedad Argentina de Protozoología; Resistencia; Argentina; 2018; 78-79CONICET DigitalCONICETenginfo:eu-repo/semantics/reference/hdl/https://ri.conicet.gov.ar/handle/11336/115949info:eu-repo/semantics/altIdentifier/url/https://protozoologia.org.ar/wp-content/uploads/XXXReunionAnualSAP.pdfInternacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T15:24:16Zoai:ri.conicet.gov.ar:11336/201729instacron: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:34982025-10-15 15:24:16.996CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
| dc.title.none.fl_str_mv |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| title |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| spellingShingle |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs Wehrendt, Diana Patricia TAQMAN Diagnostics Trypanosoma cruzi Reservoirs |
| title_short |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| title_full |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| title_fullStr |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| title_full_unstemmed |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| title_sort |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
| dc.creator.none.fl_str_mv |
Wehrendt, Diana Patricia Gómez Bravo, Andrea Pech May, Angélica del Rosario Ramsey, Janine Cura, Carolina Inés Curto, Maria de Los Angeles Abril, M. Guhl, F. Schijman, Alejandro Gabriel |
| author |
Wehrendt, Diana Patricia |
| author_facet |
Wehrendt, Diana Patricia Gómez Bravo, Andrea Pech May, Angélica del Rosario Ramsey, Janine Cura, Carolina Inés Curto, Maria de Los Angeles Abril, M. Guhl, F. Schijman, Alejandro Gabriel |
| author_role |
author |
| author2 |
Gómez Bravo, Andrea Pech May, Angélica del Rosario Ramsey, Janine Cura, Carolina Inés Curto, Maria de Los Angeles Abril, M. Guhl, F. Schijman, Alejandro Gabriel |
| author2_role |
author author author author author author author author |
| dc.contributor.none.fl_str_mv |
Lucero, Raul Horacio |
| dc.subject.none.fl_str_mv |
TAQMAN Diagnostics Trypanosoma cruzi Reservoirs |
| topic |
TAQMAN Diagnostics Trypanosoma cruzi 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 |
A major question of epidemiological relevance in Chagas disease studies is to understand the dynamics ofT.cruzi infection in sylvatic and domestic transmission cycles and to trace the origins of (re)emerging casesin areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity and molecularapproaches, such as the polymerase chain reaction may ll in this gap, provided that a standardizedmethod can be developed and validated.We have developed two duplex Real Time PCR assays for sensitive detection of T.cruzi satellite or minicirclerepetitive sequences in blood samples from mammal reservoirs, incorporating an internal amplicationstandard that allows distinction of false negative PCR ndings due to inadequate conditions of storageand transport of samples, DNA degradation during nucleic acid purication and/or inhibition of PCR byinterfering substances present in the sample. The housekeeping gene that encodes the interphotoreceptorretinoid-binding protein (IRBP) has been selected as internal standard, because it is highly conserved amongall mammal species and its usefulness as a DNA integrity control was previously reported in a conventionalPCR protocol. Based on the alignment of the IRBP sequence available for several domestic and wild reservoirspecies, we designed primers and a TaqMan probe to a highly conserved region. The analytical sensitivitywas 0.01 par. eq/mL and 0.1 par.eq/mL for satDNA/IRBP duplex and kDNA/IRBP duplex, respectively, astested with two series of canine blood spiked with known concentrations of Silvio X10 (Tc I) and CL-Brener(Tc VI) cultured epimastigotes. The assays were evaluated in DNA extracts from blood samples of 87 wildand 147 domestic animals. Our DNA integrity control worked well for wild reservoir species including smallrodents (Akodon toba, Galea leucoblephara, Rattus rattus), opossums (Didelphis virginiana, D. marsupialis),bats (Tadarida brasiliensis, Promops nasutus, Desmodus rotundus) and other mammals such as the skunk,viscacha, wildcat, brown brocket deer, hare and Pampas fox. Mean Ct values for IRBP amplication variedamong species between 24 and 33. For domestic reservoirs, IRBP amplied in dog, cat, cow, sheep, goat,horse and mule specimens with mean Ct values between 23 and 25. In T.cruzi infected cases the assaysallowed quantication of parasitic loads.Our results promote the use of these methods for rapid and accurate screening of T.cruzi infection in dierentspecies of reservoirs. 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: Pech May, Angélica del Rosario. Instituto Nacional de Salud Pública; México. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ramsey, Janine. Instituto Nacional de Salud Pública; México Fil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Curto, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Abril, M.. Fundación Mundo Sano; Argentina Fil: Guhl, F.. Universidad de los Andes; 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 XXX Reunión Anual de la Sociedad Argentina de Protozoología Resistencia Argentina Sociedad Argentina de Protozoología |
| description |
A major question of epidemiological relevance in Chagas disease studies is to understand the dynamics ofT.cruzi infection in sylvatic and domestic transmission cycles and to trace the origins of (re)emerging casesin areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity and molecularapproaches, such as the polymerase chain reaction may ll in this gap, provided that a standardizedmethod can be developed and validated.We have developed two duplex Real Time PCR assays for sensitive detection of T.cruzi satellite or minicirclerepetitive sequences in blood samples from mammal reservoirs, incorporating an internal amplicationstandard that allows distinction of false negative PCR ndings due to inadequate conditions of storageand transport of samples, DNA degradation during nucleic acid purication and/or inhibition of PCR byinterfering substances present in the sample. The housekeeping gene that encodes the interphotoreceptorretinoid-binding protein (IRBP) has been selected as internal standard, because it is highly conserved amongall mammal species and its usefulness as a DNA integrity control was previously reported in a conventionalPCR protocol. Based on the alignment of the IRBP sequence available for several domestic and wild reservoirspecies, we designed primers and a TaqMan probe to a highly conserved region. The analytical sensitivitywas 0.01 par. eq/mL and 0.1 par.eq/mL for satDNA/IRBP duplex and kDNA/IRBP duplex, respectively, astested with two series of canine blood spiked with known concentrations of Silvio X10 (Tc I) and CL-Brener(Tc VI) cultured epimastigotes. The assays were evaluated in DNA extracts from blood samples of 87 wildand 147 domestic animals. Our DNA integrity control worked well for wild reservoir species including smallrodents (Akodon toba, Galea leucoblephara, Rattus rattus), opossums (Didelphis virginiana, D. marsupialis),bats (Tadarida brasiliensis, Promops nasutus, Desmodus rotundus) and other mammals such as the skunk,viscacha, wildcat, brown brocket deer, hare and Pampas fox. Mean Ct values for IRBP amplication variedamong species between 24 and 33. For domestic reservoirs, IRBP amplied in dog, cat, cow, sheep, goat,horse and mule specimens with mean Ct values between 23 and 25. In T.cruzi infected cases the assaysallowed quantication of parasitic loads.Our results promote the use of these methods for rapid and accurate screening of T.cruzi infection in dierentspecies of reservoirs. |
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2018 |
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2018 |
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Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs; XXX Reunión Anual de la Sociedad Argentina de Protozoología; Resistencia; Argentina; 2018; 78-79 CONICET Digital CONICET |
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