Incorporación de Uracilo ADN glicosilasa / dUTPs en la reacción de PCR anidada para detectar <i>Plasmodium falciparum</i> y <i>Plasmodium vivax</i>: una estrategia para reducir el riesgo de contaminación

Carlos Alejandro Herrera-Sandoval; Tatiana María Lopera-Mesa

doi:10.17533/udea.acbi.v42n113a06

Authors

Carlos Alejandro Herrera-Sandoval Universidad de Antioquia https://orcid.org/0000-0001-9371-5555
Tatiana María Lopera-Mesa Universidad de Antioquia https://orcid.org/0000-0002-9401-2779

DOI:

https://doi.org/10.17533/udea.acbi.v42n113a06

Keywords:

DNA contamination, Malaria, Plasmodium sp., Polymerase Chain Reaction, Uridine triphosphate

Abstract

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.

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Author Biographies

Carlos Alejandro Herrera-Sandoval, Universidad de Antioquia

Grupo Malaria, Universidad de Antioquia, Medellín, Colombia. Estudiante de Maestría, Corporación de Ciencias Básicas Biomédicas, Universidad de Antioquia, Colombia.

Tatiana María Lopera-Mesa, Universidad de Antioquia

Grupo Malaria, Universidad de Antioquia, Medellín, Colombia. Docente e investigadora, Facultad de Medicina, Universidad de Antioquia, Colombia.

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