Trypanosoma cruziGenotypes ofInsect Vectorsand Patients with Chagas of Chile Studied by Means ofCytochrome bGene Sequencing, Minicircle Hybridization, and Nuclear Gene Polymorphisms

Abstract

Fifty-six Trypanosoma cruzi stocks from Chile and neighboring countries and different hosts, humans, and Triatoma infestans and Mepraia sp., vectors of domiciliary and natural environments were characterized by using three molecular markers. These were cytochrome b (Cyt b) gene sequencing, minicircle DNA blotting, and hybridization with five genotype-specific DNA probes and nuclear analysis of 1f8 and gp72 by polymerase chain reaction-restriction fragment length polymorphism. The results with all three molecular markers are concordant, with minor limitations, grouping T. cruzi stocks into four discrete typing units (DTUs) (TcI, TcII, TcV, and TcVI). TcI and TcII stocks were heterogeneous. TcI and TcII stocks were clustered in two main subgroups determined by Cyt b gene sequencing and minicircle hybridization. However, TcV and TcVI stocks were homogeneous and not differentiated by Cyt b gene sequencing or minicircle DNA hybridization. The discriminatory power and limitations of the molecular markers are discussed, as well as the distribution of the four DTUs in the domiciliary and sylvatic transmission cycles of Chile and the limitations of each marker for molecular epidemiological studies performed with T. cruzi stocks rather than the analysis of direct T. cruzi samples from natural hosts.

Differential Pattern of Infection of Sylvatic Nymphs and Domiciliary Adults of Triatoma infestans with Trypanosoma cruzi Genotypes in Chile

“…6,7 Moreover, a recent study showed that genotyping a large number of T. cruzi stocks and clones by means of hybridization was concordant with other methods, as cytochrome b gene sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 8 Similarly, genotyping results obtained with hybridization tests of T. cruzi patient samples from Colombia were concordant with the markers miniexon, 24α ribosomal RNA, and cytochrome oxidase subunit II genes. 9 Trypanosoma cruzi has been classified into six genetic subdivisions or lineages: TcI-TcVI.…”

cytb as a New Genetic Marker for Differentiation of Prototheca Species

“…Compared with nuclear DNA, mitochondrial DNA offers certain advantageous characteristics, including a high rate of evolution, limited proneness to recombination, haploidy (single-locus genes), and high copy number per cell (59,67). Among many different mitochondrion-based markers, the cytb gene, coding for cytochrome b, a transmembrane protein forming the core of the mitochondrial cytochrome bc1 complex of the respiratory chain, has been one of the most widely exploited and has been successfully used in resolving phylogenetic relationships across the broad spectrum of eukaryotic lineages at a variety of taxonomic levels (68)(69)(70)(71). This is because the cytb gene is variable enough to allow discrimination between even very closely related species and conservative enough to define relationships above the species level.…”

Effects of mammal host diversity and density on the infection level of Trypanosoma cruzi in sylvatic kissing bugs

“…The PCR method is highly specific (Junqueira et al, 2005), and the sensitivity limit has been estimated as 0.01 parasite equivalents/PCR assay (Arenas et al, 2012). The amplification reaction for faecal and blood samples was performed in triplicate with oligonucleotides 121 (5 ′ -AAA TAA TGT ACG G (T/G) GAG ATG CAT GA-3 ′ ) and 122 (5 ′ GGG TTC GATTGG GGT TGG TGT-3), which anneal to the four constant regions in mini-circles of T. cruzi (Botto-Mahan et al, 2005a).…”