title: Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces.
abstract: Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and sub-assemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.
authors.affiliation: Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia. amy.asher@mq.edu.au
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Asher
authors.forename: Liette S
authors.initials: LS
authors.lastname: Waldron
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Power
mesh_terms: D005243:Feces / Q000469:parasitology*; D016829:Giardia lamblia / Q000235:genetics / Q000302:isolation & purification*; D005873:Giardiasis / Q000175:diagnosis* / Q000469:parasitology; D005969:Glutamate Dehydrogenase / Q000235:genetics; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D015800:Protozoan Proteins; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S; D005969:Glutamate Dehydrogenase