Development of a protocol for soil transmitted helminths DNA extraction from feces by combining commercially available solutions

Abstract

ABSTRACTBackgroundOne of the main challenges for the mass introduction of molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effect of various pretreatment procedures on the efficiency of removing PCR inhibitors and extractingToxocara canisDNA from feces.Methodology and main resultsIn the first part of the work, we evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer of a commercial kit) on the integrity of Toxocara eggs using microscopy and the efficiency of DNA extraction using PCR. Our results showed that Toxocara eggs were most effectively destroyed using the bead beating procedure, while the effect of enzymes and freeze-heat cycles did not lead to significant destruction of the eggs or the release of Toxocara DNA. In the second part of the work, we evaluated the effect of prewashes with 0.1% Tween-20 solution and the use of commercial concentrators on DNA extraction from fecal samples contaminated withT. caniseggs. We have shown that the use of commercial concentrators in combination with sample washing can significantly increase the DNA yield and reduce PCR inhibition.ConclusionsA bead beating procedure for 30 minutes at a shaking frequency of 50 Hz was sufficient to completely destroy theToxocara caniseggs. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The use of a commercial concentrator followed by washing with a 0.1% Tween-20 solution can significantly increase the yield of STHs DNA and reduce PCR inhibition.AUTHOR SUMMARYDNA-based techniques are increasingly being used for the diagnosis of intestinal helminth infections in both clinical and research laboratories. However, extracting DNA from intestinal worm eggs in feces remains a challenge because the very tough eggshell protects their DNA. In addition, feces contain inhibitors that can interfere with test results, and these must be removed during DNA extraction. In the present study, we assessed the effect of different STHs egg destruction methods, as well as concentration and washing procedures for fecal samples, on the PCR test results. We have shown that bead beating is the most effective and sufficient method for the complete destruction of helminth eggs. In addition, we have shown that parasite concentrators significantly increase the sensitivity of the PCR-based test.