Caracterização do gene 18S rRNA em parasitos do grupo Apicomplexa: uma abordagem aplicada à seleção de marcadores moleculares

Abstract

The Apicomplexa group comprises protozoa that cause globally distributed diseases such as malaria, toxoplasmosis, or opportunistic intestinal disorders. The major protozoa of medical importance are usually identified by light microscopy, which makes it difficult the precise diagnosis especially in cases where parasitemia is low. In this context, the present work had as objective to develop an alternative molecular method, that allows the identification of a wide variety of protozoa of the group Apicomplexa. Thus, a primer system was developed for use in a semi-nested PCR (Polymerase Chain Reaction). The investigated target for primer design was the 18S rRNA gene, as it is a widely used target for species identification and screening in biodiversity studies. From the sequence analysis and characterization of potential formation of secondary structures, sets of primers that anneal in conserved regions flanking variable regions of the gene were designed. The efficiency of each primer set was evaluated by in silico PCR. A set of primers that can generate ~ 166 amplicons was selected, to be used in a nested reaction. These primers can be used to discriminate genus and species of Apicomplexa by difference in agarose gel size and by sequencing. The proposed method was validated in vitro and its efficiency in the identification of some protozoan species of medical interest was confirmed. After additional validation steps, this method can be used for initial screening in cases of suspected es and also for determination of different species of parasites.