2026-11-132026-11-132025-06-27HABER, Giovanna Zandonadi. Diagnóstico molecular de infecção por Leishmania (Leishmania) chagasi em soro de cães domésticos e animais silvestres provenientes de área endêmica de leishmaniose visceral no estado do Pará. Orientador: Patrícia Karla Santos Ramos. 2025. 107 f. Dissertação (Mestrado em Biologia Parasitária na Amazônia) - Centro de Ciências Biológicas e da Saúde, Universidade do Estado do Pará, Belém, 2025.https://portaldaposgraduacao.uepa.br/handle/riuepa/461AbertoLeishmaniose VisceraBiologia MolecularDiagnóstico molecularVisceral LeishmaniasisMolecular BiologyMolecular diagnosisDissertaçãoCiências BiológicasCiências Biológicas::ParasitologiaCiências Biológicas::Parasitologia::Protozoologia de ParasitosCiências Biológicas::Parasitologia::Protozoologia de Parasitos::Protozoologia Parasitária AnimalCiências Biológicas::BioquímicaCiências Biológicas::Bioquímica::Biologia MolecularVisceral Leishmaniasis (VL), classified as a neglected tropical disease, represents a serious public health concern closely linked to poverty and poor sanitary conditions. Transmitted by phlebotomine sandflies and having as vertebrate hosts not only humans but also dogs and wild animals, its diagnosis requires precise laboratory methods, which are essential for effective treatment. In this context, the present study aimed to molecularly diagnose Leishmania (L.) chagasi infection in serum samples from dogs and wild animals in the state of Pará, through the application of indirect ELISA for IgG detection and qPCR. A total of 85 samples were analyzed —60 from dogs and 25 from wild animals— collected from both endemic areas (Barcarena/PA) and non-endemic areas (Metropolitan Region of Belém/PA). The indirect ELISA was performed using previously standardized antigens, while in the qPCR assay, primers and a probe were used, targeting the kDNA minicircle region of the parasite. The standardization of DNA extraction and qPCR was successful, with good reproducibility and a limit of detection (LOD) at approximately Cq=34. The optimal reagent concentrations were established as 500nM for primers, 200nM for probe, and a 1/10⁶ dilution of the positive control. The assay demonstrated high sensitivity (100%), efficiency of 110.4%, and an R² value of 0.996. However, limitations in specificity (2.17%) were observed, attributed to the suboptimal positioning of the TaqMan®-MGB probe and the presence of single nucleotide polymorphisms (SNPs), which led to cross-reactions with the negative control and other Leishmania and Trypanosoma species. The comparison with the indirect ELISA revealed minimal agreement between the methods due to their methodological differences. In response to this, a new set of oligonucleotides (designated the GIO set) was developed, targeting the kDNA minicircle region to improve specificity. Despite the specificity limitations, the method showed potential to detect infections even in samples with low parasite loads, highlighting the need for further refinement of the oligonucleotides used.PPG-BPA - Fisiopatologia humana e experimental de processos infecciosos