title: 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.
abstract: 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.
authors.affiliation: Synlab Group, Microbiology Service, Quirón Madrid University Hospital, Diego de Velázquez 1, 28223 Pozuelo de Alarcón, Madrid, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Paulos
authors.affiliation: Veterinary Faculty, Alfonso X El Sabio University, Avenida Universidad 1, 28691 Villanueva de la Cañada, Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Mateo
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Aida
authors.initials: A
authors.lastname: de Lucio
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Hernández-de Mingo
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Begoña
authors.initials: B
authors.lastname: Bailo
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Saugar
authors.affiliation: Livestock Laboratory, Regional Government of Álava, Ctra. de Azua 4, 01520 Vitoria-Gasteiz, Spain.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Cardona
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Fuentes
authors.affiliation: Synlab Group, Microbiology Service, Quirón Madrid University Hospital, Diego de Velázquez 1, 28223 Pozuelo de Alarcón, Madrid, Spain; European University of Madrid, Tajo s/n, 28670 Villaviciosa de Odón, Madrid, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Mateo
authors.affiliation: Parasitology Service, National Centre for Microbiology, Carlos III Health Institute, Ctra. Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain. Electronic address: dacarmena@isciii.es.
authors.forename: David
authors.initials: D
authors.lastname: Carmena
mesh_terms: D000818:Animals; D003457:Cryptosporidiosis / Q000175:diagnosis*; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000302:isolation & purification*; D004747:Entamoeba / Q000235:genetics / Q000302:isolation & purification; D004749:Entamoebiasis / Q000175:diagnosis*; D005243:Feces / Q000469:parasitology*; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification; D005873:Giardiasis / Q000175:diagnosis*; D006801:Humans; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic
keywords: Commercial kit; Cryptosporidium; DNA; Entamoeba dispar; Entamoeba histolytica; Enteric; Extraction; Giardia duodenalis; Protozoan parasites; Purification