Usefulness of new DNA extraction procedure for PCR technique in species identification of Entamoeba isolates

In this study we have developed a modified CLARK and DIAMOND (1992, 1993) method using the polymerase chain reaction to amplify amoebic ribosomal RNA genes that allow either specific detection of Entamoeba histolytica s. str. or amoeba species identification. DNA was isolated from cultured protozoa or from stool samples (cysts and/or trophozoites). Cultures of xenic or axenic material (also stool samples) were treated with Easy Genomic DNA Prep or Genomic DNA Prep Plus (A&A Biotechnology, Poland) for DNA isolation. With these new procedures, DNA can be obtained effectively and with high degree of purity. 13 amoeba strains were investigated. Three strains produced an 876 bp fragment with Psp5 and Psp3 primers (specific product for E. histolytica s. str). Three strains gave a specific 876 bp product for E. dispar with NPsp5 and NPsp3 primers. Two strains were E. moshkovskii as identified by KpnI digestion of PCR products obtained with RD5 and RD3 primers. Four strains (one of them was cultured in two laboratories) were E. invadens as identified by Hinfl or HhaI digestion of PCR products obtained with RD5 and RD3 primers. Characteristic RFLP patterns with these enzymes was also obtained for E. terrapinae and Blastocystis hominis. This RFLP analysis show significant promise as a medical diagnostic tool particularly useful for species identification.