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

• Published in: Parasites & vectors Vol. 6; no. 1; p. 363
• Main Authors: de Sá, Amanda Regina Nichi, Steindel, Mário, Demeu, Lara Maria Kalempa, Lückemeyer, Débora Denardin, Grisard, Edmundo Carlos, Neto, Quirino Alves de Lima, de Araújo, Silvana Marques, Toledo, Max Jean de Ornelas, Gomes, Mônica Lúcia
• Format: Journal Article
• Language: English
• Published: England Springer-Verlag 23.12.2013; BioMed Central Ltd; BioMed Central
• Subjects: Base Sequence; Cytochrome oxidase; cytochrome-c oxidase; digestive system; DNA; DNA sequencing; DNA, Protozoan - genetics; Electron Transport Complex IV - genetics; Electron Transport Complex IV - metabolism; epidemiological studies; Epidemiology; Gene Expression Regulation, Enzymologic; Genes; Genetic aspects; Genetic polymorphisms; Genomics; hosts; Infections; Leishmania; Molecular Sequence Data; Nucleotide sequencing; Parasites; polymerase chain reaction; Polymorphism, Restriction Fragment Length; Protozoa; restriction fragment length polymorphism; Species Specificity; Triatominae; Trypanosoma; Trypanosoma cruzi; Trypanosoma cruzi - enzymology; Trypanosoma cruzi - genetics; Trypanosoma cruzi - isolation & purification; Trypanosoma rangeli; Trypanosoma rangeli - enzymology; Trypanosoma rangeli - genetics; Trypanosoma rangeli - isolation & purification; Latin America; Brazil
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/1756-3305-6-363

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.