DNA EXTRACTION OF GASTROINTESTINAL HELMINTHS EGGS OF FECAL SAMPLES USING DIFFERENT PROTOCOLS

Abstract

Helminths found in the gastrointestinal tract pose significant challenges in managing and controlling animal health issues on farms. To effectively control and prevent these parasites, it is necessary to have access to epidemiological and structural information about them. Molecular approaches, specifically DNA sequence analysis, are commonly employed for identifying and quantifying different nematode species. However, extracting pure genomic DNA from internal parasites is challenging due to the presence of a hard eggshell, which hinders access to the parasitic DNA and complicates the extraction process. In this study, we assessed the performance of three DNA extraction methods: TRIZOLE method, TRIZOLE with sonication and the commercially available GeneJET Genomic DNA Purification kit. Initially, fecal samples were collected from four sheep and subsequently homogenized and centrifuged. Sugar was added to the samples to form sheather sugar flotation solution, followed by another round of centrifugation to confirm the presence of parasite eggs in all fecal samples through microscopic analysis. Once egg presence was confirmed, DNA extraction was performed using the TRIZOLE method, TRIZOLE with sonication and GeneJET Genomic DNA Purification kit. Gel analysis was conducted to determine the presence or absence of DNA bands in the extracted samples. The results revealed that no DNA bands were observed when using the TRIZOLE method, whereas DNA smears were formed in the case of sonication method. In contrast, clear DNA bands were observed in samples where DNA extraction was performed using the GeneJET Genomic DNA Purification kit. Based on these findings, it can be concluded that the kit method was more effective for extracting DNA from gastrointestinal nematode eggs compared to the other three methods.