Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood |
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| Affiliation: | 1. USDA/ARS Foreign Animal Disease Research Unit, Plum Island Animal Disease Center, P.O. Box 848, Greenport, NY 11944, USA;2. Instituto de Biotecnología, INTA, Buenos Aires, Argentina;3. Museo Argentino de Ciencias Naturales Bernardino Rivadavia, CONICET, Buenos Aires, Argentina;4. FMD Virology Department, OIE FMD Reference Laboratory, DLA, Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Dirección de Laboratorio Animal, Argentina;5. University of Minnesota, Department of Veterinary Population Medicine, College of Veterinary Medicine, Saint Paul, MN, USA;1. Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile;2. Programa de Microbiología, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile;1. Department of Pediatrics, National School of Tropical Medicine, Baylor College of Medicine and Texas Children''s Hospital, Houston, TX, USA;2. Department of Tropical Medicine, Vector-Borne Infectious Disease Research Center, Tulane University, School of Public Health and Tropical Medicine, New Orleans, LA, USA;3. Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Autonomous University of Yucatan (UADY), Merida, Yucatan, Mexico;1. Grupo de Investigaciones Microbiológicas – UR (GIMUR), Programa de Biología, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia;2. Grupo de Investigaciones Biológicas de la Orinoquia, Unitrópico, Yopal, Colombia;1. Unidade Integrada Sesi Senai, Niquelândia, Goiás, Brazil;2. Universidade Federal do Paraná, Department of Bioprocess Engineering and Biotechnology, Molecular Biology Laboratory, Curitiba, Paraná, Brazil;3. Universidade Federal do Pará, Centre for Valorisation of Amazonian Bioactive Compounds, Belém, Pará, Brazil;4. Universidade Federal do Paraná, Department of Biochemistry, Curitiba, Paraná, Brazil |
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| Abstract: | The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a major public health problem in Latin America. This parasite has a complex population structure comprised by six or seven major evolutionary lineages (discrete typing units or DTUs) TcI-TcVI and TcBat, some of which have apparently resulted from ancient hybridization events. Because of the existence of significant biological differences between these lineages, strain characterization methods have been essential to study T. cruzi in its different vectors and hosts. However, available methods can be laborious and costly, limited in resolution or sensitivity. In this study, a new genotyping strategy by real-time PCR to identify each of the six DTUs in clinical blood samples have been developed and evaluated.Two nuclear (SL-IR and 18S rDNA) and two mitochondrial genes (COII and ND1) were selected to develop original primers. The method was evaluated with eight genomic DNA of T. cruzi populations belonging to the six DTUs, one genomic DNA of Trypanosoma rangeli, and 53 blood samples from individuals with chronic Chagas disease. The assays had an analytical sensitivity of 1–25 fg of DNA per reaction tube depending on the DTU analyzed. The selectivity of trials with 20 fg/μL of genomic DNA identified each DTU, excluding non-targets DTUs in every test. The method was able to characterize 67.9% of the chronically infected clinical samples with high detection of TcII followed by TcI.With the proposed original genotyping methodology, each DTU was established with high sensitivity after a single real-time PCR assay. This novel protocol reduces carryover contamination, enables detection of each DTU independently and in the future, the quantification of each DTU in clinical blood samples. |
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