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

Background One 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 extracting Toxocara canis DNA from feces. Methodology and main results In 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 with T. canis eggs. We have shown that the use of commercial concentrators in combination with sample washing can significantly increase the DNA yield and reduce PCR inhibition. Conclusions A bead beating procedure for 30 minutes at a shaking frequency of 50 Hz was sufficient to completely destroy the Toxocara canis eggs. 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.