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

Published online by Cambridge University Press:  15 March 2012

A. J. ASHER ,
L. S. WALDRON  and
M. L. POWER
A. J. ASHER*
Affiliation:
Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
L. S. WALDRON
Affiliation:
Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
M. L. POWER
Affiliation:
Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia
*
*Corresponding author: Tel: +612 9850 9259. Fax: +612 9850 8245. E-mail: [email protected]
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Summary

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.

Keywords

Giardia duodenalisT-RFLPgdhrapid identificationclinical samples
Type
Research Article
Information
Parasitology , Volume 139 , Issue 8 , July 2012 , pp. 1005 - 1013
Copyright
Copyright © Cambridge University Press 2012

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