Giardia intestinalis: DNA extraction approaches to improve PCR results |
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| Authors: | Babaei Zahra Oormazdi Hormozd Rezaie Sasan Rezaeian Mostafa Razmjou Elham |
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| Affiliation: | aDepartment of Parasitology and Mycology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran;bDepartment of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran;cDepartment of Parasitology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran |
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| Abstract: | Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol. |
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| Keywords: | Flagellate protozoa DNA extraction method Glass beads PCR Glutamate Dehydrogenase gene |
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