Dados do Trabalho

Título

Comparison of two techniques: RFLP with sequencing, after PCR-ITS1 to identify Leishmania species causing American Tegumentary Leishmaniasis

Introdução

American tegumentary leishmaniasis(ATL) is an infectious disease caused by the protozoan Leishmania and the gold standard laboratory diagnosis is the parasitological technique, but it cannot identify species, therefore, molecular diagnosis is indicated for the identification of species. Polymerase Chain Reaction(PCR) is a molecular diagnosis and the ITS-1 region of the ribosomal RNA gene has an adequate number of polymorphisms for distinction at least at the species level.The RFLP (restriction fragment length polymerase) after PCR/ITS-1 is used to identify the different Leishmania species based on the cleavage of DNA molecules by the restriction enzyme HaeIII, which generates fragments of different sizes. Sequencing is also used after PCR/ITS-1, therefore the products obtained from PCR/ITS1 were subjected to the method described by Sanger.

Objetivo(s)

The aim of our study was to compare RFLP with sequencing after PCR-ITS-1 to identify Leishmania species or even Leishmania subgenus that cause ATL.

Material e Métodos

The procedures were: 1.DNA was extracted from 211 samples obtained from biopsies of patients with suspected tegumentary leishmaniasis; 2.After DNA extraction, it was submitted to PCR-ITS-1 (LITSR and L5.8S) under the conditions described by Godoy et al.(2020); 3.Fifty-nine (59) amplified products obtained from PCR-ITS1 were submitted to digestion by the PCR-RFLP technique, using HaeIII FastDigest®; 4.Thirty-seven (37) amplified products obtained from PCR-ITS1 were subjected to Sanger Sequencing. For the genomic sequencing of the purified products, the BigDye® Terminator Cycle Sequencing Kit was used.

Resultados e Conclusão

The results obtained were: 40.28% of the samples (85/211) amplified in PCR for the ITS1 gene (320 pb); 45.7% (27/59) presented fragments with cutting patterns corresponding to the RFLP and 54.2% (32/59) had no cuts/fragments after performing RFLP; 83.73% (31/37) of the samples showed species identification by sequencing, making it possible to define 6 species of Leishmania. Six samples did not show significant identification or similarity. Our conclusion is that although the RFLP technique is cheaper than sequencing, it cannot define species or even subgenus in the same quantities as the sequencing.Therefore, the sequencing of PCR-ITS1 products is indicated and has epidemiological values, as it allows the identification at level of Leishmania species, that occur in some localities of Brazil affected by ATL.

Palavras-chave

American tegumentary leishmaniasis, PCR, ITS-1 gene, RFLP, Sequencing