Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism

A. J. Asher, L.S. Waldron, M. L. Power

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages1005-1013
Number of pages9
JournalParasitology
Volume139
Issue number8
DOIs
Publication statusPublished - Jul 2012

Fingerprint

Giardia lamblia
Restriction Fragment Length Polymorphisms
restriction fragment length polymorphism
DNA Sequence Analysis
sequence analysis
Bovine Immunodeficiency Virus
Giardia
Intestinal Diseases
Glutamate Dehydrogenase
South Australia
New South Wales
glutamate dehydrogenase
digestive system diseases
Protozoa
Parasites
Fluorescence
fluorescence

Cite this

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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.",
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Rapid identification of Giardia duodenalis assemblages in NSW using terminal-restriction fragment length polymorphism. / Asher, A. J.; Waldron, L.S.; Power, M. L.

In: Parasitology, Vol. 139, No. 8, 07.2012, p. 1005-1013.

Research output: Contribution to journalArticleResearchpeer-review

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