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PLoS Negl Trop Dis. 2015 May 19;9(5):e0003765. doi: 10.1371/journal.pntd.0003765. eCollection 2015 May.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

Author information

1
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.
2
Instituto de Medicina Regional, Universidad Nacional del Nordeste, Resistencia, Chaco, Argentina.
3
Laboratoire Hospitalier et Universitaire-CH Andrée Rosemon, Cayenne, French Guiana, France.
4
Laboratorio Biología Celular, Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico.
5
Servicio de Medicina Interna, Hospital Politécnico LA FE, Valencia, Spain.
6
Servicio de Microbiología, Hospital Universitario y Politécnico LA FE, Valencia, Spain.
7
Laboratorio de Investigación en Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, Lima, Peru.
8
Department of Biology, University of Minnesota Duluth, Duluth, Minnesota, United States of America.
9
Laboratorio de Parasitología, Instituto Nacional de Laboratorios en Salud, Ministerio de Salud y Deportes de Bolivia, La Paz, Bolivia.
10
Southwest National Primate Research Center and Department of Genetics, Texas Biomedical Research Institute, San Antonio, Texas, United States of America.
11
Instituto de Biología de la Altura, Universidad Nacional de Jujuy, Jujuy, Argentina.
12
Epidemiología e Infectología Clínica, Hospital Universitario Fundación Favaloro, Buenos Aires, Argentina.
13
Sección Infectología, Servicio de Clínica Médica, Hospital Italiano, Buenos Aires, Argentina.
14
Institut de Recherche pour le Développement and University Paris Descartes, UMR 216, Mother and Child Facing Tropical Diseases, Paris, France.
15
Parasitologia Médica e Biologia de Vetores, Área de Patologia, Faculdade de Medicina, Universidade de Brasilia, Brasilia DF, Brazil.
16
Laboratorio de Eco-Epidemiología, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
17
Centro Regional de Investigación en Salud Pública, Instituto Nacional de Salud Pública, Tapachula, Chiapas, Mexico.
18
Drugs and Neglected Diseases Initiative, Genève, Switzerland.
19
Pan American Health Organization (PAHO), World Health Organization (WHO), Washington, D.C., United States of America.
20
Institute of Biotechnology, Molecular Parasitology Group, University of Granada, Granada, Spain.

Abstract

BACKGROUND:

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).

METHODS/PRINCIPAL FINDINGS:

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.

CONCLUSIONS/SIGNIFICANCE:

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.

PMID:
25993316
PMCID:
PMC4437652
DOI:
10.1371/journal.pntd.0003765
[Indexed for MEDLINE]
Free PMC Article

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