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Diagn Microbiol Infect Dis. 2014 Jan;78(1):53-8. doi: 10.1016/j.diagmicrobio.2013.09.008. Epub 2013 Sep 28.

Simplified protocol for DNA extraction and amplification of 2 molecular markers to detect and type Giardia duodenalis.

Author information

1
Laboratory of Environmental Parasitology, Department of Basic Health Sciences, State University of Maringá, Maringá, Paraná 87020-900, Brazil.
2
Laboratory of Environmental Parasitology, Department of Basic Health Sciences, State University of Maringá, Maringá, Paraná 87020-900, Brazil. Electronic address: criscolli@yahoo.com.br.
3
Integrated College at Campo Mourão, Paraná, Brazil.

Abstract

We evaluated the ability of 3 kits: QIAmp® DNA stool mini kit (Qiagen, Hilden, Germany), PureLink PCR Purification®, and PureLink™ Genomic DNA® (Invitrogen, Carlsbad, CA, USA) for DNA extraction, and of 2 molecular markers (heat shock protein [HSP] and β-giardin genes) for detection and genotyping of Giardia duodenalis stool samples. The detection and typing limits of the markers were determined by the DNA concentration of trophozoites and cysts and were tested in 26 clinical samples. Of the 3 kits tested, the PureLink PCR Purification gave the best results when tested with clinical samples with low, intermediate, and high numbers of cysts. The DNA extracted from trophozoites and cysts was diluted successively in 1:2 ratios until it was no longer possible to observe the amplified product in polyacrylamide gel. Similarly, a suspension of cysts was diluted until no cysts were observed, and then the DNA was extracted. The amount of DNA of trophozoites and cysts for the typing of the parasite was smaller for the HSP marker than for β-giardin. Combined use of both markers allowed us to detect DNA of Giardia in parasitologically positive samples in a higher percentage (75%) than the results obtained for each marker and in 1 parasitologically negative sample, indicating that this combination increased the potential to accurately detect and genotype this parasite. We also concluded that the HSP marker has a higher limit of detection and typing than the β-giardin marker and that the DNA extraction method tested for G. duodenalis is simpler and more efficient than those that are currently in use and can be applied on a large scale.

KEYWORDS:

Detection limit; Giardia; Heat shock protein; Molecular typing; β giardin

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