Comparison of four dna extraction methods from stool Samples for the detection of intestinal parasites while Optimizing molecular tools at jimma university Molecular biology laboratory

Abstract

Intestinal helminths and protozoan parasites are among the most prevalent parasites causing a significant morbidity and mortality to humans in developing countries. Commonly used diagnostic methods for these parasites rely mainly on microscopical examination of stool samples for the identification of helminth eggs and protozoan trophozoites and cysts. However, this method is not efficient enough to differentiate morphologically indistinguishable interspecies and strains of parasites. Thus, the present study was aimed to evaluate different DNA extraction methods for the identification and further advanced molecular based studies of intestinal parasites in terms of their effectiveness, cost and time. Moreover, the present study was also aimed at optimizing basic molecular biology techniques and tools to be used in newly established molecular biology laboratory at Jimma University. We compared and evaluated two commercially purchased DNA extraction kits (QIAamp stool mini kit and DNeasy blood and tissue kit) with and without their modifications of beat beating. The comparison of these four methods were done by collecting and examining 195 stool samples from schoolchildren attending primary schools in Jimma Town where high prevalence of soiltransmitted helminth (STH) infections has been reported recently. These stool samples were categorized in to three groups of 10 based on the intensity of infection as (high, low and no) eggs per gram of stool (EPG) for each of the three STH (Ascaris lumbricoides, Trichuris trichiura, and hookworms) and subjected to four different DNA extraction methods. Finally, the agreements of the results were compared using nested PCR followed by electrophoresis and visualization of DNA bands using UV trans-illuminator. The comparison of these different DNA extraction methods was also performed for Giardia duodenalis. Since we don’t have a recommended gold standard diagnostic or DNA extraction method from stool samples for diagnosis of intestinal parasites, we have used measure of agreement "kappa" using SPSS version 20 for the evaluation of effectiveness of different DNA extraction methods. Finally, DNeasy blood and tissue kit with bead beating method is appeared to be the most effective DNA extraction method in terms of DNA yield, cost and time. Therefore, we would like recommending this kit as relatively effective DNA extraction methods for soil transmitted helminthes and G. duodenalis in resource poor regions like our country Ethiopia. Moreover, we recommend further evaluation of those kits for other parasites.