Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma rangeli and Trypanosoma cruzi

Abstract

BackgroundThe parasites Trypanosoma rangeli and Trypanosoma cruzi share vectors and hosts over a wide geographical area in Latin America. In this study, we propose a single molecular approach for simultaneous detection and typing of T. rangeli and T. cruzi.MethodsA restriction fragment length polymorphism analysis of the mitochondrial cytochrome oxidase II gene (COII-RFLP) using enzyme AluI and different amounts of DNA from the major genetic groups of T. rangeli and T. cruzi (KP1+/KP1- and DTU-I/DTU-II) was carried out. The same marker was tested on the other T. cruzi DTUs (DTU-III to DTU-VI) and on DNA extracted from gut contents of experimentally infected triatomines.ResultsThe COII PCR generates a ~400 bp fragment, which after digestion with AluI (COII-RFLP) can be used to distinguish T. rangeli from T. cruzi and simultaneously differentiate the major genetic groups of T. rangeli (KP1+ and KP1-) and T. cruzi (DTU-I and DTU-II). The COII-RFLP generated bands of ~120 bp and ~280 bp for KP1+, whereas for KP1- no amplicon cleavage was observed. For T. cruzi, digestion of COII revealed a ~300 bp band for DTU-I and a ~250 bp band for DTU-II. For DTU-III to DTU-VI, COII-RFLP generated bands ranging from ~310 to ~330 bp, but the differentiation of these DTUs was not as clear as the separation between DTU-I and DTU-II. After AluI digestion, a species-specific fragment of ~80 bp was observed for all DTUs of T. cruzi. No cross-amplification was observed for Leishmania spp., T. vivax or T. evansi.ConclusionsThe COII-RFLP allowed simultaneous detection and typing of T. rangeli and T. cruzi strains according to their major genetic groups (KP1+/KP1- and DTU-I/DTU-II) in vitro and in vivo, providing a reliable and sensitive tool for epidemiological studies in areas where T. rangeli and T. cruzi coexist.

Identification of bat trypanosomes from Minas Gerais state, Brazil, based on 18S rDNA and Cathepsin-L-like targets

“…COII RFLP-PCR can differentiate T. cruzi DTUs and T. rangeli KP1+ of KP1− and co-infection between these parasites (de Sá et al 2013). In this study 14 samples amplified fragments with sizes compatible with the fragment obtained from TcII and EM437 strain amplified non-specific band.…”

Surveillance and genotype characterization of zoonotic trypanosomatidae in Didelphis marsupialis in two endemic sites of rural Panama

“…In our study, the higher T. cruzi infection rate in opossums compared to previous reports may be a true increase in prevalence, or due to improved molecular detection methods. Molecular assays are highly effective and sensitive for the detection of T. rangeli infection ( Vallejo et al, 1999 ; Pavia et al, 2007 ; de Sá et al, 2013 ). In addition, these methods allow the evaluation of the genetic diversity of parasite populations, thus enabling a better understanding of the infection behavior among a given reservoir population.…”

“…PCR were performed using S35/S36 primers that amplify a 330 bp segment of the T. cruzi variable region minicircle and the primer pairs TrF/R2 that amplify a 620 bp fragment of T. rangeli snoRNA-c11 gene ( Vallejo et al, 1999 ; Pavia et al, 2007 ). T. cruzi and T. rangeli positive samples were typed by a miniexon gene-based approach ( Fernandes et al, 2001 ) and a PCR-RFLP approach targeting the COII gene ( de Sá et al, 2013 ), respectively. T. cruzi DTUs were detected by a real time PCR strategy based on the amplification of a set of the specific markers SL-IR (TcI-TcIII), COII (TcII-TcIV), ND1 (TcV) and 18S rDNA (TcVI) that in combination discriminate all T. cruzi DTUs ( Muñoz-San Martín et al, 2017 ).…”

Investigation regarding the presence of Trypanosoma cruzi in triatomines and humans in rural households in the State of Paraná, southern Brazil

“…For XC, an aliquot of 250 μL of the IC was inoculated into 2.5 mL of LIT (Liver infusion tryptose) culture medium, with and without antibiotic (6.6 mg/mL of penicillin) (Bronfen et al, 1989;Bisugo et al, 1998). Finally, the remaining IC was added to 500 µL of 70 °GL ethyl alcohol and stored at -20 ºC until DNA extraction (Sá et al, 2013;Sá et al, 2016). The remaining 10 specimens of P. megistus from Janiópolis were received without life and were stored in bottles containing a 70 °GL ethyl alcohol solution, not allowing the performance of FE and XC.…”

“…The material previously obtained by abdominal compression of these insects was submitted to microscopic analysis by the DVAS technicians. Thus, it was not possible to remove the rectal ampoule for insect dissection and, for these specimens, DNA extraction was performed with the macerate of the entire insect (Sá et al, 2013).…”