Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs
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 lac...
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ftconicet:oai:ri.conicet.gov.ar:11336/201729 2023-10-09T21:55:34+02:00 Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs 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 Lucero, Raul Horacio Internacional application/pdf application/vnd.openxmlformats-officedocument.wordprocessingml.document http://hdl.handle.net/11336/201729 eng eng Sociedad Argentina de Protozoología info:eu-repo/semantics/reference/hdl/https://ri.conicet.gov.ar/handle/11336/115949 info:eu-repo/semantics/altIdentifier/url/https://protozoologia.org.ar/wp-content/uploads/XXXReunionAnualSAP.pdf http://hdl.handle.net/11336/201729 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 info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ TAQMAN Diagnostics Trypanosoma cruzi Reservoirs https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 info:eu-repo/semantics/publishedVersion info:eu-repo/semantics/conferenceObject info:ar-repo/semantics/documento de conferencia Reunión Book ftconicet 2023-09-24T20:02:23Z 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 ... Book Rattus rattus CONICET Digital (Consejo Nacional de Investigaciones Científicas y Técnicas) |
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Open Polar |
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CONICET Digital (Consejo Nacional de Investigaciones Científicas y Técnicas) |
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ftconicet |
language |
English |
topic |
TAQMAN Diagnostics Trypanosoma cruzi Reservoirs https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
spellingShingle |
TAQMAN Diagnostics Trypanosoma cruzi Reservoirs https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 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 Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic reservoirs |
topic_facet |
TAQMAN Diagnostics Trypanosoma cruzi Reservoirs https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
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 ... |
author2 |
Lucero, Raul Horacio |
format |
Book |
author |
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_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_sort |
Wehrendt, Diana Patricia |
title |
Development of duplex TaqMan PCR assays for detection and quantification of Trypanosoma cruzi infection in wild and domestic 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 |
publisher |
Sociedad Argentina de Protozoología |
url |
http://hdl.handle.net/11336/201729 |
op_coverage |
Internacional |
genre |
Rattus rattus |
genre_facet |
Rattus rattus |
op_relation |
info:eu-repo/semantics/reference/hdl/https://ri.conicet.gov.ar/handle/11336/115949 info:eu-repo/semantics/altIdentifier/url/https://protozoologia.org.ar/wp-content/uploads/XXXReunionAnualSAP.pdf http://hdl.handle.net/11336/201729 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 |
op_rights |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
_version_ |
1779319523695919104 |