TY - JOUR A1 - Paulos Viñas, Silvia AU - Mateo, Marta AU - Lucio, Aída de AU - Hernández de Mingo, Marta AU - Bailo, Begoña AU - Saugar, José M. AU - Cardona, Guillermo A. AU - Fuentes, Isabel AU - Mateo Barrientos, María AU - Carmena, David T1 - Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp Y1 - 2016 SN - 01677012 UR - http://hdl.handle.net/11268/6101 AB - High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n = 29), giardiasis (n = 47) and amoebiasis by Entamoeba histolytica (n = 3) or E. dispar (n = 10) and apparently healthy subjects (n = 24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56 °C were proven more efficient for the release of DNA from Cryptosporidium oocysts. KW - Protozoan parasites KW - DNA KW - ADN KW - Genética humana KW - Biotecnología KW - Genética LA - eng ER -