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Memorias del Instituto de Investigaciones en Ciencias de la Salud

versão On-line ISSN 1812-9528


CHAVES, Enmanuel Céspedes et al. Standardization of PCR-RFLP technique of the ITS-1 region for Leishmania (L.) infantum molecular characterization in canine samples. Mem. Inst. Investig. Cienc. Salud [online]. 2016, vol.14, n.3, pp.24-33. ISSN 1812-9528.

Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. These parasites infect to mammalian hosts, including canines that have been implicated as reservoirs of the parasite. The aim of this research was to standardize the technique of PCR-RFLP after amplification of the ITS1 region of Leishmania (Leishmania) infantum, as a useful tool for detection and molecular characterization. Promastigotes from culture and biopsies from dogs with visceral Leishmaniasis previously diagnosed by the Centro Antirrábico Nacional. The ITS1 region of the genomic DNA of Leishmania sp. was amplified using LITSR and L5,8S primers. The technique ITS1 PCR-RFLP applied, allowed the detection of Leishmania (L.) infantum in 10/10 of the isolates from parasites maintained in NNN culture medium, in 10/18 samples from spleen and 10/18 samples from popliteal lymph node. Optimal reaction conditions were 0.2 mM dNTPs, 0.1 pmol of each primer and 1U of Taq polymerase. The sensitivity of PCR was 3 ng/µL in isolates of parasites from NNN culture medium and 60 ng/µL in biopsy samples while the specificity was 100% for the detection of DNA of Leishmania sp. The restriction enzyme Hae III determined fragments of 184, 72 and 55 bp., which were specific to Leishmania (L.) infantum. The marker used is reliable for the detection and characterization of Leishmania sp. in dogs from endemic areas, which could be useful to verify the species of parasites circulating among animals.

Palavras-chave : Leishmania infantum; dogs; ITS1 (Internal Transcribed Spacer 1).

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