Dataset Information

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences.

ABSTRACT: We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.

SUBMITTER: Chiurillo MA

PROVIDER: S-EPMC193880 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences.

Chiurillo Miguel Angel MA Crisante Gladys G Rojas Agustina A Peralta Andreina A Dias Manuel M Guevara Palmira P Añez Néstor N Ramírez José Luis JL

Clinical and diagnostic laboratory immunology 20030901 5

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was rob ...[more]

PMID: 12965903

Similar Datasets

Project description:BACKGROUND:A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. RESULTS:The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. CONCLUSIONS:This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

Project description:BackgroundTrypanosoma conorhini and Trypanosoma rangeli, like Trypanosoma cruzi, are kinetoplastid protist parasites of mammals displaying divergent hosts, geographic ranges and lifestyles. Largely nonpathogenic T. rangeli and T. conorhini represent clades that are phylogenetically closely related to the T. cruzi and T. cruzi-like taxa and provide insights into the evolution of pathogenicity in those parasites. T. rangeli, like T. cruzi is endemic in many Latin American countries, whereas T. conorhini is tropicopolitan. T. rangeli and T. conorhini are exclusively extracellular, while T. cruzi has an intracellular stage in the mammalian host.ResultsHere we provide the first comprehensive sequence analysis of T. rangeli AM80 and T. conorhini 025E, and provide a comparison of their genomes to those of T. cruzi G and T. cruzi CL, respectively members of T. cruzi lineages TcI and TcVI. We report de novo assembled genome sequences of the low-virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E ranging from ~ 21-25 Mbp, with ~ 10,000 to 13,000 genes, and for the highly virulent and hybrid T. cruzi CL we present a ~ 65 Mbp in-house assembled haplotyped genome with ~ 12,500 genes per haplotype. Single copy orthologs of the two T. cruzi strains exhibited ~ 97% amino acid identity, and ~ 78% identity to proteins of T. rangeli or T. conorhini. Proteins of the latter two organisms exhibited ~ 84% identity. T. cruzi CL exhibited the highest heterozygosity. T. rangeli and T. conorhini displayed greater metabolic capabilities for utilization of complex carbohydrates, and contained fewer retrotransposons and multigene family copies, i.e. trans-sialidases, mucins, DGF-1, and MASP, compared to T. cruzi.ConclusionsOur analyses of the T. rangeli and T. conorhini genomes closely reflected their phylogenetic proximity to the T. cruzi clade, and were largely consistent with their divergent life cycles. Our results provide a greater context for understanding the life cycles, host range expansion, immunity evasion, and pathogenesis of these trypanosomatids.

Dataset Information

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences.

Publications

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets