Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces

Amy J. Asher, Liette S. Waldron, Michelle L. Power

Research output: Contribution to journalArticleResearchpeer-review

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 subassemblage 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.

LanguageEnglish
Pages853-858
Number of pages6
JournalParasitology Research
Volume110
Issue number2
DOIs
Publication statusPublished - Feb 2012

Fingerprint

Giardia lamblia
Feces
feces
Polymerase Chain Reaction
Glutamate Dehydrogenase
loci
glutamate dehydrogenase
DNA
Bovine Immunodeficiency Virus
Molecular Diagnostic Techniques
Genotype
Small Ribosome Subunits
Genes
Giardia
sampling
Ribosomal RNA
genes
genotype
human diseases
diagnostic techniques

Cite this

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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 subassemblage 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.",
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Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces. / Asher, Amy J.; Waldron, Liette S.; Power, Michelle L.

In: Parasitology Research, Vol. 110, No. 2, 02.2012, p. 853-858.

Research output: Contribution to journalArticleResearchpeer-review

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