Resumo

HERRERA SANDOVAL, Carlos Alejandro  e  LOPERA MESA, Tatiana María. Uracil-DNA-glycosilase system (UDG-dUTPs) incorporation in nested PCR reaction for Plasmodium falciparum and Plasmodium vivax detection: a strategy for reducing risk of contamination. Actu Biol [online]. 2020, vol.42, n.113, e4.  Epub 07-Maio-2021. ISSN 0304-3584.  https://doi.org/10.17533/udea.acbi/v42n113a01.

Polymerase chain reaction (PCR) has been used in research and diagnostics as method to confirm malaria infections. Due to its high sensitivity, this method is susceptible to amplicon contamination, when high number of samples are processed, increasing the risk of false positives. This study aimed at evaluating the incorporation of Uracil DNA glycosylase-dUTPs (UDG-dUTPs) system to nested PCR reaction (nPCR) for Plasmodium falciparum and Plasmodium vivax, as a strategy to prevent contamination in new reactions. The DNA of P. falciparum 3D7 strain and a clinical sample confirmed with P. vivax were used in all reactions. The effect of replacing dTTPs by dUTPs in nPCR reaction was evaluated, and the effect of this modification on the limit of detection was verified. The degradative action of the UDG was evaluated in artificially contaminated reactions with amplicons. The amount of contaminating DNA that was degraded by a unit of UDG was quantified. Replacement of dTTPs by dUTPs did not affect Taq polymerase performance, however, a slight decrease in the analytical sensitivity of nPCR when using dUTPs was observed. UDG was able to exclusively degrade the contaminants without affecting the amplification of the native DNA. One unit of UDG was able to completely degrade contaminating DNA up to approximately 6 pg/μL. UDG-dUTPs system can prevent contamination to improve molecular malaria diagnosis.

Palavras-chave : DNA contamination; malaria; Plasmodium sp.; Polymerase Chain Reaction; uridine triphosphate.

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