Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples
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, selectivit...
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2015
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_19352727_v9_n5_p_Cura http://hdl.handle.net/20.500.12110/paper_19352727_v9_n5_p_Cura |
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paper:paper_19352727_v9_n5_p_Cura2023-06-08T16:31:54Z Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples 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 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. 2015 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_19352727_v9_n5_p_Cura http://hdl.handle.net/20.500.12110/paper_19352727_v9_n5_p_Cura |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples |
| topic_facet |
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 |
| description |
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. |
| title |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples |
| title_short |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples |
| title_full |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples |
| title_fullStr |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples |
| title_full_unstemmed |
Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples |
| title_sort |
multiplex real-time pcr assay using taqman probes for the identification of trypanosoma cruzi dtus in biological and clinical samples |
| publishDate |
2015 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_19352727_v9_n5_p_Cura http://hdl.handle.net/20.500.12110/paper_19352727_v9_n5_p_Cura |
| _version_ |
1768543204179705856 |