Comparison and evaluation of four methods for extracting DNA from Giardia duodenalis cysts for PCR targeting the tpi gene.

Abstract

Giardia duodenalis is an intestinal flagellated protozoan and the common cause of gastrointestinal diseases in human. This parasite can be seen in two different forms in its life cycle including as cyst and trophozoite. Due to presence of resistant cyst wall, DNA extraction inhibitors along with artifact in stool specimens, this study was performed aiming to evaluate four methods for DNA extraction from G. duodenalis cysts. Seventy G. duodenalis positive stool specimens that were confirmed by light microscope were included in this study. All stool samples were concentrated using four layered discontinuous sucrose flotation technique (0.5, 0.75, 1, and 1.5 M) and single-layered sucrose solution (0.85 M). The isolated cysts were then subjected to DNA extraction by four methods. To remove the artifacts, the extracted DNA were evaluated by PCR. The results of the present study showed the high level of optical density (OD) in the method I (P < 0.01) with the following steps; Giardia cysts plus crushed cover glass were vortexed. Then, the samples were boiled and then followed by freeze-thaw cycles, yet this method yielded the lowest concentration. Furthermore, the highest concentration were observed in the method II (P <0.01) with the following steps; Giardia cysts plus crushed cover glass and TAE buffer were mixed and then shaken, followed by boiling. Based on the results of the present study, using crushed cover glass, boiling and freeze-thaw cycles can be effective in destruction of G. duodenalis cyst wall and have enough efficiency for extracting DNA from G. duodenalis cysts.

• Authors: Akram Sepahvand, Nader Pestehchian, Hossein Ali Yousefi, Reza Pahlavan Gharehbaba
• Journal: Journal of parasitic diseases : official organ of the Indian Society for Parasitology (J Parasit Dis) Vol. 41 Issue 1 Pg. 263-267 (Mar 2017) ISSN: 0971-7196 [Print] India
• PMID: 28316423 (Publication Type: Journal Article)