title: Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis.
abstract: We evaluated the ability of 3 kits: QIAmp® DNA stool mini kit (Qiagen, Hilden, Germany), PureLink PCR Purification®, and PureLink™ Genomic DNA® (Invitrogen, Carlsbad, CA, USA) for DNA extraction, and of 2 molecular markers (heat shock protein [HSP] and β-giardin genes) for detection and genotyping of Giardia duodenalis stool samples. The detection and typing limits of the markers were determined by the DNA concentration of trophozoites and cysts and were tested in 26 clinical samples. Of the 3 kits tested, the PureLink PCR Purification gave the best results when tested with clinical samples with low, intermediate, and high numbers of cysts. The DNA extracted from trophozoites and cysts was diluted successively in 1:2 ratios until it was no longer possible to observe the amplified product in polyacrylamide gel. Similarly, a suspension of cysts was diluted until no cysts were observed, and then the DNA was extracted. The amount of DNA of trophozoites and cysts for the typing of the parasite was smaller for the HSP marker than for β-giardin. Combined use of both markers allowed us to detect DNA of Giardia in parasitologically positive samples in a higher percentage (75%) than the results obtained for each marker and in 1 parasitologically negative sample, indicating that this combination increased the potential to accurately detect and genotype this parasite. We also concluded that the HSP marker has a higher limit of detection and typing than the β-giardin marker and that the DNA extraction method tested for G. duodenalis is simpler and more efficient than those that are currently in use and can be applied on a large scale.
authors.affiliation: Laboratory of Environmental Parasitology, Department of Basic Health Sciences, State University of Maringá, Maringá, Paraná 87020-900, Brazil.
authors.forename: Carla Fernanda
authors.initials: CF
authors.lastname: Uda-Shimoda
authors.affiliation: Laboratory of Environmental Parasitology, Department of Basic Health Sciences, State University of Maringá, Maringá, Paraná 87020-900, Brazil. Electronic address: criscolli@yahoo.com.br.
authors.forename: Cristiane Maria
authors.initials: CM
authors.lastname: Colli
authors.affiliation: Integrated College at Campo Mourão, Paraná, Brazil.
authors.forename: Mariana Felgueira
authors.initials: MF
authors.lastname: Pavanelli
authors.affiliation: Laboratory of Environmental Parasitology, Department of Basic Health Sciences, State University of Maringá, Maringá, Paraná 87020-900, Brazil.
authors.forename: Ana Lúcia
authors.initials: AL
authors.lastname: Falavigna-Guilherme
authors.affiliation: Laboratory of Environmental Parasitology, Department of Basic Health Sciences, State University of Maringá, Maringá, Paraná 87020-900, Brazil.
authors.forename: Mônica Lúcia
authors.initials: ML
authors.lastname: Gomes
mesh_terms: D003598:Cytoskeletal Proteins / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification*; D005819:Genetic Markers; D060005:Genotyping Techniques / Q000379:methods*; D016829:Giardia lamblia / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005873:Giardiasis / Q000175:diagnosis* / Q000469:parasitology; D006360:Heat-Shock Proteins / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity
chemical_list: D003598:Cytoskeletal Proteins; D016054:DNA, Protozoan; D005819:Genetic Markers; D006360:Heat-Shock Proteins; D011933:Reagent Kits, Diagnostic
keywords: Detection limit; Giardia; Heat shock protein; Molecular typing; β giardin