Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence

Autores
Muñoz Calderon, Arturo Alejandro; Silva Gomess, Natalia Lins; Apodaca, Sofia; Alarcón de Noya, Belkisyolé; Díaz Bello, Zoraida; Quintino Souza, Leticia Rocha; Tavares Costa, Alexandre Dias; Britto, Constança; Moreira, Otacilio Cruz; Schijman, Alejandro Gabriel
Año de publicación
2021
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.
Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; Brasil
Fil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; Venezuela
Fil: Díaz Bello, Zoraida. Universidad Central de Venezuela; Venezuela
Fil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; Brasil
Fil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; Brasil
Fil: Britto, Constança. Fundación Oswaldo Cruz; Brasil
Fil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; Brasil
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
Materia
REAL TIME PCR
PARASITIC LOAD QUANTIFICATION
MOLECULAR DIAGNOSIS
SYNTHETIC OLIGONUCLEOTIDE
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/153492
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/153492
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequenceMuñoz Calderon, Arturo AlejandroSilva Gomess, Natalia LinsApodaca, SofiaAlarcón de Noya, BelkisyoléDíaz Bello, ZoraidaQuintino Souza, Leticia RochaTavares Costa, Alexandre DiasBritto, ConstançaMoreira, Otacilio CruzSchijman, Alejandro GabrielREAL TIME PCRPARASITIC LOAD QUANTIFICATIONMOLECULAR DIAGNOSISSYNTHETIC OLIGONUCLEOTIDEhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; BrasilFil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; VenezuelaFil: Díaz Bello, Zoraida. Universidad Central de Venezuela; VenezuelaFil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; BrasilFil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; BrasilFil: Britto, Constança. Fundación Oswaldo Cruz; BrasilFil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; BrasilFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaAmerican Society of Investigative Pathology2021-05-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/153492Muñoz Calderon, Arturo Alejandro; Silva Gomess, Natalia Lins; Apodaca, Sofia; Alarcón de Noya, Belkisyolé; Díaz Bello, Zoraida; et al.; Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence; American Society of Investigative Pathology; Journal Of Molecular Diagnostics; 23; 5; 4-5-2021; 521-5311525-1578CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://linkinghub.elsevier.com/retrieve/pii/S1525157821000283info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmoldx.2021.01.007info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:18:02Zoai:ri.conicet.gov.ar:11336/153492instacron: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:34982025-09-29 10:18:03.154CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
title Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
spellingShingle Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
Muñoz Calderon, Arturo Alejandro
REAL TIME PCR
PARASITIC LOAD QUANTIFICATION
MOLECULAR DIAGNOSIS
SYNTHETIC OLIGONUCLEOTIDE
title_short Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
title_full Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
title_fullStr Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
title_full_unstemmed Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
title_sort Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence
dc.creator.none.fl_str_mv Muñoz Calderon, Arturo Alejandro
Silva Gomess, Natalia Lins
Apodaca, Sofia
Alarcón de Noya, Belkisyolé
Díaz Bello, Zoraida
Quintino Souza, Leticia Rocha
Tavares Costa, Alexandre Dias
Britto, Constança
Moreira, Otacilio Cruz
Schijman, Alejandro Gabriel
author Muñoz Calderon, Arturo Alejandro
author_facet Muñoz Calderon, Arturo Alejandro
Silva Gomess, Natalia Lins
Apodaca, Sofia
Alarcón de Noya, Belkisyolé
Díaz Bello, Zoraida
Quintino Souza, Leticia Rocha
Tavares Costa, Alexandre Dias
Britto, Constança
Moreira, Otacilio Cruz
Schijman, Alejandro Gabriel
author_role author
author2 Silva Gomess, Natalia Lins
Apodaca, Sofia
Alarcón de Noya, Belkisyolé
Díaz Bello, Zoraida
Quintino Souza, Leticia Rocha
Tavares Costa, Alexandre Dias
Britto, Constança
Moreira, Otacilio Cruz
Schijman, Alejandro Gabriel
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv REAL TIME PCR
PARASITIC LOAD QUANTIFICATION
MOLECULAR DIAGNOSIS
SYNTHETIC OLIGONUCLEOTIDE
topic REAL TIME PCR
PARASITIC LOAD QUANTIFICATION
MOLECULAR DIAGNOSIS
SYNTHETIC OLIGONUCLEOTIDE
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.
Fil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Silva Gomess, Natalia Lins. Fundación Oswaldo Cruz; Brasil
Fil: Apodaca, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina
Fil: Alarcón de Noya, Belkisyolé. Universidad Central de Venezuela; Venezuela
Fil: Díaz Bello, Zoraida. Universidad Central de Venezuela; Venezuela
Fil: Quintino Souza, Leticia Rocha. Fundación Oswaldo Cruz; Brasil
Fil: Tavares Costa, Alexandre Dias. Fundación Oswaldo Cruz; Brasil
Fil: Britto, Constança. Fundación Oswaldo Cruz; Brasil
Fil: Moreira, Otacilio Cruz. Fundación Oswaldo Cruz; Brasil
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
description Accurate diagnostics tools and surrogate markers of parasitological response to treatment are priority needs for management of Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a Satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (Tc I) and CL-Brener (Tc VI) stocks similar LOD95 values (0.903 (0.745-1.497) and 0.667 (CI 0.113-3.927) copy numbers/μL, respectively), when synthetic DNA was the standard for quantification, thus allowing direct comparison of loads in samples infected with different DTUs. This standard curve was evaluated in 205 samples from 38 acute oral and 19 chronic CD patients from different geographical areas infected with different genotypes, including samples obtained during treatment follow-up, and high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase III clinical trials, to follow-up patients under treatment or at risk of reactivation and in experimental models using different parasite strains.
publishDate 2021
dc.date.none.fl_str_mv 2021-05-04
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/153492
Muñoz Calderon, Arturo Alejandro; Silva Gomess, Natalia Lins; Apodaca, Sofia; Alarcón de Noya, Belkisyolé; Díaz Bello, Zoraida; et al.; Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence; American Society of Investigative Pathology; Journal Of Molecular Diagnostics; 23; 5; 4-5-2021; 521-531
1525-1578
CONICET Digital
CONICET
url http://hdl.handle.net/11336/153492
identifier_str_mv Muñoz Calderon, Arturo Alejandro; Silva Gomess, Natalia Lins; Apodaca, Sofia; Alarcón de Noya, Belkisyolé; Díaz Bello, Zoraida; et al.; Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using a synthetic satellite unit DNA sequence; American Society of Investigative Pathology; Journal Of Molecular Diagnostics; 23; 5; 4-5-2021; 521-531
1525-1578
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://linkinghub.elsevier.com/retrieve/pii/S1525157821000283
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmoldx.2021.01.007
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Society of Investigative Pathology
publisher.none.fl_str_mv American Society of Investigative Pathology
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
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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score 13.070432

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