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 singlelayered 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.

Evaluation of Three Protocols of DNA Extraction for Detection of Giardia duodenalis in Human Fecal Specimens

“…Using the sucrose flotation tech-nique, freeze-thawing, and adding glass beads have demonstrated the successful approaches in extraction and subsequent molecular identification of G. duodenalis (4,22,38). In this study, comparison of the efficiency of 3 extraction protocols on PCR fragments have shown the Phenol-Chloroform Isoamyl alcohol technique as the most effective method, especially in terms of increased quantity of the extracted DNA.…”

“…Molecular PCR based assays have been successfully applied for characterization of Giardia genotypes using the small subunit ribosomal RNA (SSU rRNA); beta-giardin (bg); elongation factor 1-α; glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes, that are extremely different in genetic variability (1,5,(22)(23)(24)(25)(26)(27). Among these markers the partial gdh, bg, tpi, and SSU rRNA genes are mostly used for identification of this parasite at the assemblage level (6,28).…”

“…Various numbers of in-house methods such as the traditional Phenol-Chloroform Isoamyl alcohol method and commercial kits like QIAamp DNA stool minikit (QIAGEN, Germany) have been assessed (4,5,8,22,29,30) for DNA extraction of G. duodenalis from stool specimens. These protocols, which are based on mechanical, chemical, and/or enzymatic lyses have been enabled researchers to extract, purify, and subsequently evaluate DNA of Giardia in molecular analysis.…”

Comparación de métodos de extracción de ADN de Giardia spp. medidos por PCR convencional

“…Asimismo, se encontró que realizar un sólo pretratamiento de choque térmico (métodos IXB y XB) resultó insuficiente para lograr la amplificación de más del 20% del marcador gdh. En concordancia a lo reportado por Sepahvand et al (18) ; quienes sugieren el uso de perlas de vidrio, para producir un efecto mecánico y mejorar la eficiencia del protocolo de extracción para recuperar mayor cantidad de ADN. Esto se evaluó al aplicar el método III, recuperando 4,17 ng/uL de ADN logrando una amplificación del 100% de las muestras para el marcador bg, pero con dificultades para detectar el marcador gdh, motivo por el que se consideró que es importante adicionar algún pretratamiento enzimático para mejorar la recuperación de ácidos nucleicos.…”

“…Las muestras se guardaron a -20 °C hasta ser procesadas. Método III (18) Pretratamiento mecánico 1: Uso de perlas de vidrio. Pretratamiento choque térmico 2: (100 °C -nitrógeno líquido) por tres minutos repetidos en seis ciclos.…”