NESTED PCR ASSAY FOR DETECTION OF LEISHMANIA GENUS SSU RNAR GENE
Autor(es): Eloiza Teles Caldart, Fernanda Pinto Ferreira, Hannah Lia Ettiene Peruch Lemos dos Santos, Regina Mitsuka-Breganó, Roberta Lemos Freire, Italmar Teodorico Navarro, João Luis Garcia
NESTED PCR ASSAY FOR DETECTION OF LEISHMANIA GENUS SSU RNAR GENE
» Área de pesquisa: DOENÇAS VETORIAIS
» Instituição: UNIVERSIDADE ESTADUAL DE LONDRINA
» Agência de fomento e patrocinadores: CNPq
Leishmaniasis is a parasitic disease that is rather difficult to diagnose. This is due to the generally low number of parasites found in clinical samples. The aim of this study was to standardize a nested-PCR targeted to a conserved central part of the SSU RNAr gene to detect Leishmania genus. The two pair of primers used in this work were R332, R221 (603bp-long product) and R333, R222 (393bp-long product) based on Van Eys et al. (1992). Modifications were made in R221 (R221.1) and R222 (R222.1) using an alignment with more recent sequences (2008-2011) and to balance the anneling temperature of the primers. Optimal annealing temperatures were tested in a gradient thermocycler; for the first reaction we tested from 54°C to 60°C and to the second 55°C to 65°C. Different concentrations of MgCL2 (1,5 to 2,5mM) were also tested, the detection limit was evaluated with a L. (Viannia) amazonensis strain. The final protocol for the first reaction was 94°C for 5min followed by 30 cycles of 94°C for 30s, 60°C for 30s, 72°C for 30s, followed by a final extension at 72°C 5min; using 20pmol of primers R221.1 and R332, 0.6UI of Platinum Taq DNA Polymerase, 1mM for each DNTP, 1µL of DNA solution, PCR Buffer 10X with 1,5mM MgCL2 for a final volume of 25µL. The final protocol for the second reaction had 65°C for the anneling temperature, the primers were R222.1 and R333 and 1µL of amplified DNA was used. The reaction sensibility was 2pg of DNA. L. (V.) braziliensis and L. infantum strains were used in the nested-PCR and the two were positive for the assay. The nested PCR reported here is a sensitive tool for leishmania genus diagnosis.
