Artículo

Cura, C.I.; Duffy, T.; Lucero, R.H.; Bisio, M.; Péneau, J.; Jimenez-Coello, M.; Calabuig, E.; Gimenez, M.J.; Valencia Ayala, E.; Kjos, S.A.; Santalla, J.; Mahaney, S.M.; Cayo, N.M.; Nagel, C.; Barcán, L.; Málaga Machaca, E.S.; Acosta Viana, K.Y.; Brutus, L. (...) Schijman, A.G. "Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples" (2015) PLoS Neglected Tropical Diseases. 9(5)
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Abstract:

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. © 2015 Cura et al.

Registro:

Documento: Artículo
Título:Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples
Autor:Cura, C.I.; Duffy, T.; Lucero, R.H.; Bisio, M.; Péneau, J.; Jimenez-Coello, M.; Calabuig, E.; Gimenez, M.J.; Valencia Ayala, E.; Kjos, S.A.; Santalla, J.; Mahaney, S.M.; Cayo, N.M.; Nagel, C.; Barcán, L.; Málaga Machaca, E.S.; Acosta Viana, K.Y.; Brutus, L.; Ocampo, S.B.; Aznar, C.; Cuba Cuba, C.A.; Gürtler, R.E.; Ramsey, J.M.; Ribeiro, I.; VandeBerg, J.L.; Yadon, Z.E.; Osuna, A.; Schijman, A.G.
Filiación:Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor N. Torres”—INGEBI-CONICET, Buenos Aires, Argentina
Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Chaco, Argentina
Laboratoire Hospitalier et Universitaire-CH Andrée Rosemon, Cayenne, French Guiana, France
Laboratorio Biología Celular, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico
Servicio de Medicina Interna, Hospital Politécnico LA FE, Valencia, Spain
Servicio de Microbiología, Hospital Universitario y Politécnico LA FE, Valencia, Spain
Laboratorio de Investigación en Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, Lima, Peru
Department of Biology, University of Minnesota Duluth, Duluth, MN, United States
Laboratorio de Parasitología, Instituto Nacional de Laboratorios en Salud, Ministerio de Salud y Deportes de Bolivia, La Paz, Bolivia
Southwest National Primate Research Center and Department of Genetics, Texas Biomedical Research Institute, San Antonio, TX, United States
Instituto de Biología de la Altura, Universidad Nacional de Jujuy, Jujuy, Argentina
Epidemiología e Infectología Clínica, Hospital Universitario Fundación Favaloro, Buenos Aires, Argentina
Sección Infectología, Servicio de Clínica Médica, Hospital Italiano, Buenos Aires, Argentina
Institut de Recherche pour le Développement and University Paris Descartes, UMR 216, Mother and Child Facing Tropical Diseases, Paris, France
Parasitologia Médica e Biologia de Vetores, Área de Patologia, Universidade de Brasilia, Brasilia DF, Brazil
Laboratorio de Eco-Epidemiología, Departamento de Ecología, Genética y Evolución, Universidad de Buenos Aires, Buenos Aires, Argentina
Centro Regional de Investigación en Salud Pública, Instituto Nacional de Salud Pública, Tapachula, Chiapas, Mexico
Drugs and Neglected Diseases Initiative, Genève, Switzerland
Pan American Health Organization (PAHO), World Health Organization (WHO), Washington, DC, United States
Institute of Biotechnology, Molecular Parasitology Group, University of Granada, Granada, Spain
Palabras clave:Article; Chagas disease; congenital infection; controlled study; fluorescence; gene sequence; genotyping technique; human; human tissue; major clinical study; microbial identification; mixed infection; molecular probe; nonhuman; real time polymerase chain reaction; sensitivity and specificity; skin biopsy; Trypanosoma cruzi; adolescent; adult; bioassay; Chagas disease; child; classification; female; genetic variation; genetics; genotype; male; molecular typing; parasitology; preschool child; procedures; real time polymerase chain reaction; Trypanosoma cruzi; Adolescent; Adult; Biological Assay; Chagas Disease; Child; Child, Preschool; Coinfection; Female; Genetic Variation; Genotype; Humans; Male; Molecular Typing; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Trypanosoma cruzi
Año:2015
Volumen:9
Número:5
DOI: http://dx.doi.org/10.1371/journal.pntd.0003765
Título revista:PLoS Neglected Tropical Diseases
Título revista abreviado:PLoS. Negl. Trop. Dis.
ISSN:19352727
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_19352727_v9_n5_p_Cura

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Citas:

---------- APA ----------
Cura, C.I., Duffy, T., Lucero, R.H., Bisio, M., Péneau, J., Jimenez-Coello, M., Calabuig, E.,..., Schijman, A.G. (2015) . Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. PLoS Neglected Tropical Diseases, 9(5).
http://dx.doi.org/10.1371/journal.pntd.0003765
---------- CHICAGO ----------
Cura, C.I., Duffy, T., Lucero, R.H., Bisio, M., Péneau, J., Jimenez-Coello, M., et al. "Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples" . PLoS Neglected Tropical Diseases 9, no. 5 (2015).
http://dx.doi.org/10.1371/journal.pntd.0003765
---------- MLA ----------
Cura, C.I., Duffy, T., Lucero, R.H., Bisio, M., Péneau, J., Jimenez-Coello, M., et al. "Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples" . PLoS Neglected Tropical Diseases, vol. 9, no. 5, 2015.
http://dx.doi.org/10.1371/journal.pntd.0003765
---------- VANCOUVER ----------
Cura, C.I., Duffy, T., Lucero, R.H., Bisio, M., Péneau, J., Jimenez-Coello, M., et al. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. PLoS. Negl. Trop. Dis. 2015;9(5).
http://dx.doi.org/10.1371/journal.pntd.0003765