Detección molecular de Dientamoeba fragilis en heces: Eliminación de los inhibidores de la DNA polimerasa

Abstract

We report an assay for the DNA purification of Dientamoeba fragilis trophozoites by means of a commercially available spin-columns technique from frozen non-formalin-fixed human stool specimens containing high concentrations of DNA -polymerase inhibitors. This method was compared with a phenol/chloroform technique; followed both of them by an amplification assay and an electrophoresis of amplicons. A nested PCR assay was developed to allow the detection of D. fragilis DNA. The DNA extraction from D. fragilis trophozoites using a fast spin-columns procedure, yielded high-quality DNA, free of impurities and enzyme inhibitors; in contrast with the phenol/chloroform technique, where inhibition was observed when the amplification products were analyzed by agarose-gel electrophoresis. Besides, the addition of SAF (sodium acetateacetic acid-formalin) to faecal specimens, that affects the integrity of DNA and, consequently, hinders its further amplification, was confirmed. © 2007 Sociedad Chilena de Parasitología. Organo Oficial de la Federación Latinoamericana de Parasitólogos.