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

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.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

Detection, cloning, and distribution of minisatellites in some mammalian genomes.

The chromosomal distribution of minisatellites (cloned and/or detected using natural or synthetic tandem repeats) is strikingly different in man and mouse. In man, the vast majority is clustered in the terminal band of a subset of chromosome arms. Interestingly, the class of shorter tandem repeats called microsatellites is widespread along the chromosomes, suggesting that minisatellites can be created or maintained only in certain regions. In order to gain a better knowledge of these areas, we have studied a sub-telomeric cosmid from the pseudoautosomal region. Sixty kilobases of human genomic DNA starting approximately 20 kilobases from the human sex chromosomes telomere have previously been independently isolated in two cosmid clones (locus DXYS14) (Cooke et al., 1985); Rouyer et al., 1986). We have studied in more detail one of the two cosmids from this locus and found that it contains four different minisatellite structures representing 20 kilobases of the cosmid. These structures are unrelated to each other or to the minisatellite family described by Jeffreys et al. (1985). They display different degrees of polymorphism correlated with varying amounts of inner homogeneity. Combined with the previous description of an additional minisatellite (Cooke et al., 1985; Inglehearn and Cooke, 1990) in the contiguous cosmid, our observation shows that these structures may represent an important proportion of the DNA in sub-telomeric regions

Physical mapping of the holoprosencephaly critical region on chromosome 7q36

Holoprosencephaly (HPE) is a developmental field defect involving the brain and face. Cytogenetic deletions in patients with HPE have localized one of the HPE genes to chromosomal region 7q36. We have characterized the 7q deletions in thirteen HPE patients. The result is the construction of a high resolution physical map of 7q32-qter. As a first step towards cloning an HPE gene crucial for normal brain development, we have defined the HPE minimal critical region in 7q36 between D7S292 and D7S392.link_to_subscribed_fulltex