title: Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism.
abstract: Humans are infected by 2 genetic assemblages (A and B) of Giardia duodenalis, a protozoan parasite that causes gastro-intestinal disease. Sub-assemblages AI, AII, BIII and BIV are commonly identified in human cases. Detection requires amplification of G. duodenalis loci. Subsequent DNA sequencing or restriction fragment length polymorphism (RFLP) identifies sub-assemblages but is expensive (DNA sequencing) or insensitive (RFLP). This study investigated a fluorescence-based detection method, using terminal-restriction fragment length polymorphism (T-RFLP) of the glutamate dehydrogenase gene to characterize human infections. Clinical samples (n=73), positive for Giardia were collected in New South Wales, Australia, and were used to evaluate T-RFLP detection. The accuracy and sensitivity of T-RFLP detection was established by comparison to DNA sequencing and RFLP. Sub-assemblage assignment by T-RFLP identified BIV as the common subtype in N.S.W cases, whilst AI, AII and BIII were also detected. When compared to DNA sequencing and RFLP, analysis by T-RFLP was a reliable and reproducible method. Automated fluorescent detection enabled accurate sizing of restriction fragments and provided a sensitive alternative to RFLP. Discrimination of sub-assemblages by T-RFLP was comparable to DNA sequencing, but was efficient and inexpensive. The protocol described here provides a rapid and sensitive diagnostic tool for routine sample screenings in epidemiological research.
authors.affiliation: Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. amy.asher@mq.edu.au
authors.forename: A J
authors.initials: AJ
authors.lastname: Asher
authors.forename: L S
authors.initials: LS
authors.lastname: Waldron
authors.forename: M L
authors.initials: ML
authors.lastname: Power
mesh_terms: D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology; D005453:Fluorescence; D016829:Giardia lamblia / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005873:Giardiasis / Q000469:parasitology*; D005969:Glutamate Dehydrogenase / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D009517:New South Wales; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015800:Protozoan Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; D005969:Glutamate Dehydrogenase