Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays

Autores
Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro Gabriel; Gállego, Montserrat; Muñoz, Carmen
Año de publicación
2018
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Background Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.
Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Instituto de Salud Global de Barcelona; España
Fil: Ballart, Cristina. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; España
Fil: Llovet, Teresa. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu I Sant Pau; España
Fil: Roig, Carme. Hospital de la Santa Creu I Sant Pau; España
Fil: Gutiérrez, Cristina. Hospital de la Santa Creu I Sant Pau; España
Fil: Tebar, Silvia. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; España
Fil: Berenguer, Pere. Hospital de la Santa Creu I Sant Pau; España
Fil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; España
Fil: Posada, Elizabeth. Instituto de Salud Global de Barcelona; España
Fil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; España
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
Fil: Gállego, Montserrat. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; España
Fil: Muñoz, Carmen. Hospital de la Santa Creu I Sant Pau; España. Universitat Autònoma de Barcelona; España
Materia
Real time PCR
automatic DNA extraction
molecular diagnosis
Chagas disease
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:11336/79837
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/79837
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assaysAbras, AlbaBallart, CristinaLlovet, TeresaRoig, CarmeGutiérrez, CristinaTebar, SilviaBerenguer, PerePinazo, María-JesúsPosada, ElizabethGascón, JoaquimSchijman, Alejandro GabrielGállego, MontserratMuñoz, CarmenReal time PCRautomatic DNA extractionmolecular diagnosisChagas diseasehttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Background Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Instituto de Salud Global de Barcelona; EspañaFil: Ballart, Cristina. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; EspañaFil: Llovet, Teresa. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu I Sant Pau; EspañaFil: Roig, Carme. Hospital de la Santa Creu I Sant Pau; EspañaFil: Gutiérrez, Cristina. Hospital de la Santa Creu I Sant Pau; EspañaFil: Tebar, Silvia. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; EspañaFil: Berenguer, Pere. Hospital de la Santa Creu I Sant Pau; EspañaFil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; EspañaFil: Posada, Elizabeth. Instituto de Salud Global de Barcelona; EspañaFil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; EspañaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gállego, Montserrat. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; EspañaFil: Muñoz, Carmen. Hospital de la Santa Creu I Sant Pau; España. Universitat Autònoma de Barcelona; EspañaPublic Library of Science2018-04info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/79837Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; et al.; Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays; Public Library of Science; Plos One; 13; 4; 4-2018; 1-141932-6203CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0195738info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pubmed/29664973info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195738info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:04:14Zoai:ri.conicet.gov.ar:11336/79837instacron: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:34982025-09-03 10:04:14.27CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
spellingShingle Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
Abras, Alba
Real time PCR
automatic DNA extraction
molecular diagnosis
Chagas disease
title_short Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_full Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_fullStr Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_full_unstemmed Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
title_sort Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays
dc.creator.none.fl_str_mv Abras, Alba
Ballart, Cristina
Llovet, Teresa
Roig, Carme
Gutiérrez, Cristina
Tebar, Silvia
Berenguer, Pere
Pinazo, María-Jesús
Posada, Elizabeth
Gascón, Joaquim
Schijman, Alejandro Gabriel
Gállego, Montserrat
Muñoz, Carmen
author Abras, Alba
author_facet Abras, Alba
Ballart, Cristina
Llovet, Teresa
Roig, Carme
Gutiérrez, Cristina
Tebar, Silvia
Berenguer, Pere
Pinazo, María-Jesús
Posada, Elizabeth
Gascón, Joaquim
Schijman, Alejandro Gabriel
Gállego, Montserrat
Muñoz, Carmen
author_role author
author2 Ballart, Cristina
Llovet, Teresa
Roig, Carme
Gutiérrez, Cristina
Tebar, Silvia
Berenguer, Pere
Pinazo, María-Jesús
Posada, Elizabeth
Gascón, Joaquim
Schijman, Alejandro Gabriel
Gállego, Montserrat
Muñoz, Carmen
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Real time PCR
automatic DNA extraction
molecular diagnosis
Chagas disease
topic Real time PCR
automatic DNA extraction
molecular diagnosis
Chagas disease
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Background Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.
Fil: Abras, Alba. Universidad de Barcelona; España. Universidad de Girona; España. Instituto de Salud Global de Barcelona; España
Fil: Ballart, Cristina. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; España
Fil: Llovet, Teresa. Universitat Autònoma de Barcelona; España. Hospital de la Santa Creu I Sant Pau; España
Fil: Roig, Carme. Hospital de la Santa Creu I Sant Pau; España
Fil: Gutiérrez, Cristina. Hospital de la Santa Creu I Sant Pau; España
Fil: Tebar, Silvia. Universidad de Barcelona; España. Instituto de Salud Global de Barcelona; España
Fil: Berenguer, Pere. Hospital de la Santa Creu I Sant Pau; España
Fil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; España
Fil: Posada, Elizabeth. Instituto de Salud Global de Barcelona; España
Fil: Gascón, Joaquim. Instituto de Salud Global de Barcelona; España
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
Fil: Gállego, Montserrat. Instituto de Salud Global de Barcelona; España. Universidad de Barcelona; España
Fil: Muñoz, Carmen. Hospital de la Santa Creu I Sant Pau; España. Universitat Autònoma de Barcelona; España
description Background Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. Methodology/Principal findings We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. Conclusions/Significance This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.
publishDate 2018
dc.date.none.fl_str_mv 2018-04
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/11336/79837
Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; et al.; Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays; Public Library of Science; Plos One; 13; 4; 4-2018; 1-14
1932-6203
CONICET Digital
CONICET
url http://hdl.handle.net/11336/79837
identifier_str_mv Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; et al.; Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays; Public Library of Science; Plos One; 13; 4; 4-2018; 1-14
1932-6203
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1371/journal.pone.0195738
info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pubmed/29664973
info:eu-repo/semantics/altIdentifier/url/https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195738
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
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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