title: Cytochrome oxidase subunit 2 gene allows simultaneous detection and typing of Trypanosoma rangeli and Trypanosoma cruzi.
abstract: BACKGROUND
  The 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.
  
  
  METHODS
  A 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Amanda Regina Nichi
authors.initials: AR
authors.lastname: de Sá
authors.forename: Mário
authors.initials: M
authors.lastname: Steindel
authors.forename: Lara Maria Kalempa
authors.initials: LM
authors.lastname: Demeu
authors.forename: Débora Denardin
authors.initials: DD
authors.lastname: Lückemeyer
authors.forename: Edmundo Carlos
authors.initials: EC
authors.lastname: Grisard
authors.forename: Quirino Alves de Lima
authors.initials: QA
authors.lastname: Neto
authors.forename: Silvana Marques
authors.initials: SM
authors.lastname: de Araújo
authors.forename: Max Jean de Ornelas
authors.initials: MJ
authors.lastname: Toledo
authors.affiliation: Departamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá (UEM), Av, Colombo, 5790, Zona 7, CEP: 87020-900, Maringá, Paraná, Brazil. mlgomes@uem.br.
authors.forename: Mônica Lúcia
authors.initials: ML
authors.lastname: Gomes
mesh_terms: D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D023281:Genomics; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D013045:Species Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D057509:Trypanosoma rangeli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D016054:DNA, Protozoan; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV