Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples

Autores
Bontempi, Iván; Bizai, María Laura; Ortiz, Sylvia; Manattini, Silvia; Fabbro, Diana Lucrecia; Solari, Aldo; Diez, Cristina Noemí
Año de publicación
2016
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa = 0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed.
Fil: Bontempi, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina
Fil: Bizai, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Centro de Investigaciones sobre Endemias Nacionales; Argentina
Fil: Ortiz, Sylvia. Universidad de Chile; Chile
Fil: Manattini, Silvia. Provincia de Santa Fe. Hospital Central de Reconquista; Argentina
Fil: Fabbro, Diana Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Centro de Investigaciones sobre Endemias Nacionales; Argentina
Fil: Solari, Aldo. Universidad de Chile; Chile
Fil: Diez, Cristina Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
Materia
Dtu
Genotype
Hybridization
Mixed Infections
Mls-Pcr
Trypanosoma Cruzi
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:11336/78249
Acceder
id CONICETDig_6763dab880817c34a91f8ce20f41d472
oai_identifier_str oai:ri.conicet.gov.ar:11336/78249
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samplesBontempi, IvánBizai, María LauraOrtiz, SylviaManattini, SilviaFabbro, Diana LucreciaSolari, AldoDiez, Cristina NoemíDtuGenotypeHybridizationMixed InfectionsMls-PcrTrypanosoma Cruzihttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa = 0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed.Fil: Bontempi, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; ArgentinaFil: Bizai, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Centro de Investigaciones sobre Endemias Nacionales; ArgentinaFil: Ortiz, Sylvia. Universidad de Chile; ChileFil: Manattini, Silvia. Provincia de Santa Fe. Hospital Central de Reconquista; ArgentinaFil: Fabbro, Diana Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Centro de Investigaciones sobre Endemias Nacionales; ArgentinaFil: Solari, Aldo. Universidad de Chile; ChileFil: Diez, Cristina Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaElsevier Science2016-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/78249Bontempi, Iván; Bizai, María Laura; Ortiz, Sylvia; Manattini, Silvia; Fabbro, Diana Lucrecia; et al.; Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples; Elsevier Science; Infection, Genetics and Evolution; 43; 9-2016; 123-1291567-1348CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1567134816301988info:eu-repo/semantics/altIdentifier/doi/10.1016/j.meegid.2016.05.026info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:20:16Zoai:ri.conicet.gov.ar:11336/78249instacron: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:34982025-10-22 11:20:17.215CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
title Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
spellingShingle Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
Bontempi, Iván
Dtu
Genotype
Hybridization
Mixed Infections
Mls-Pcr
Trypanosoma Cruzi
title_short Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
title_full Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
title_fullStr Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
title_full_unstemmed Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
title_sort Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples
dc.creator.none.fl_str_mv Bontempi, Iván
Bizai, María Laura
Ortiz, Sylvia
Manattini, Silvia
Fabbro, Diana Lucrecia
Solari, Aldo
Diez, Cristina Noemí
author Bontempi, Iván
author_facet Bontempi, Iván
Bizai, María Laura
Ortiz, Sylvia
Manattini, Silvia
Fabbro, Diana Lucrecia
Solari, Aldo
Diez, Cristina Noemí
author_role author
author2 Bizai, María Laura
Ortiz, Sylvia
Manattini, Silvia
Fabbro, Diana Lucrecia
Solari, Aldo
Diez, Cristina Noemí
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Dtu
Genotype
Hybridization
Mixed Infections
Mls-Pcr
Trypanosoma Cruzi
topic Dtu
Genotype
Hybridization
Mixed Infections
Mls-Pcr
Trypanosoma Cruzi
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa = 0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed.
Fil: Bontempi, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Tecnología Inmunológica; Argentina
Fil: Bizai, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Centro de Investigaciones sobre Endemias Nacionales; Argentina
Fil: Ortiz, Sylvia. Universidad de Chile; Chile
Fil: Manattini, Silvia. Provincia de Santa Fe. Hospital Central de Reconquista; Argentina
Fil: Fabbro, Diana Lucrecia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Centro de Investigaciones sobre Endemias Nacionales; Argentina
Fil: Solari, Aldo. Universidad de Chile; Chile
Fil: Diez, Cristina Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina
description Different DNA markers to genotype Trypanosoma cruzi are now available. However, due to the low quantity of parasites present in biological samples, DNA markers with high copy number like kinetoplast minicircles are needed. The aim of this study was to complete a DNA assay called minicircle lineage specific-PCR (MLS-PCR) previously developed to genotype the T. cruzi DTUs TcV and TcVI, in order to genotype DTUs TcI and TcII and to improve TcVI detection. We screened kinetoplast minicircle hypervariable sequences from cloned PCR products from reference strains belonging to the mentioned DTUs using specific kDNA probes. With the four highly specific sequences selected, we designed primers to be used in the MLS-PCR to directly genotype T. cruzi from biological samples. High specificity and sensitivity were obtained when we evaluated the new approach for TcI, TcII, TcV and TcVI genotyping in twenty two T. cruzi reference strains. Afterward, we compared it with hybridization tests using specific kDNA probes in 32 blood samples from chronic chagasic patients from North Eastern Argentina. With both tests we were able to genotype 94% of the samples and the concordance between them was very good (kappa = 0.855). The most frequent T. cruzi DTUs detected were TcV and TcVI, followed by TcII and much lower TcI. A unique T. cruzi DTU was detected in 18 samples meantime more than one in the remaining; being TcV and TcVI the most frequent association. A high percentage of mixed detections were obtained with both assays and its impact was discussed.
publishDate 2016
dc.date.none.fl_str_mv 2016-09
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/11336/78249
Bontempi, Iván; Bizai, María Laura; Ortiz, Sylvia; Manattini, Silvia; Fabbro, Diana Lucrecia; et al.; Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples; Elsevier Science; Infection, Genetics and Evolution; 43; 9-2016; 123-129
1567-1348
CONICET Digital
CONICET
url http://hdl.handle.net/11336/78249
identifier_str_mv Bontempi, Iván; Bizai, María Laura; Ortiz, Sylvia; Manattini, Silvia; Fabbro, Diana Lucrecia; et al.; Simple methodology to directly genotype Trypanosoma cruzi discrete typing units in single and mixed infections from human blood samples; Elsevier Science; Infection, Genetics and Evolution; 43; 9-2016; 123-129
1567-1348
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.sciencedirect.com/science/article/pii/S1567134816301988
info:eu-repo/semantics/altIdentifier/doi/10.1016/j.meegid.2016.05.026
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
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier 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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score 12.982451

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