eng Oriental Scientific Publishing Company Biosciences Biotechnology Research Asia 0973-1245 2019-09-25 16 3 491 508 10.13005/bbra/2764 34206 article Dalia Abuljadayel 1 Ahmed Atef 1 Mohammed Al-Matary 1 Sherif Edris 1,2,3 Khalid M. Al-Ghamdi 1 Nahid H. Hajrah Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), P.O. Box 80141, Jeddah 21589, Saudi Arabia Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt Princess Al-Jawhara Al-Brahim Centre of Excellence in Research of Hereditary Disorders (PACER-HD), Faculty of Medicine, King Abdulaziz University (KAU), Jeddah, Saudi Arabia The Genome Analysis Center, Norwich Research Park, Norwich, NR4 7UH, UK The most commonly used approach for Entamoeba species differentiation up to date is the tRNA-linked STR regions of the parasite’s genome. In the present study, a new reliable, fast and easy molecular tool for species differentiation was developed. DNA was isolated from fecal samples collected from infected subjects with either Entamoeba histolytica (EH) or Entamoeba disper (ED) in Saudi Arabia. Two types of primer sets were compared in which the first targeted tRNA-linked STR regions, while the second was designed after multiple contig alignment of the two genomes using NUCmer program in aligned areas with high similarity (~90%) and difference between of ~90 bp. The selection criteria secures that designed primers should pair with both EH and ED contig sequences at homologous regions of 200-500 bp of both species except for the presence of indels that result in the recovery of amplicons of two species with different sizes. Banding patterns in the tRNA-linked STR region resulted in the occurrence of several common amplicons. We speculate that primers mismatch with regions other than the specified STR arrays of Entamoeba histolytica or Entamoeba disper with organisms other than Entamoeba existed in the fecal sample. However, the STR-based approach looked very useful in studying strain differentiation and parasite diversity. The results for the new approach complemented those of the STR-based approach, except that the latter failed to detect coinfected subjects. The new approach proved to be useful at the species level, while the tRNA-linked STR approach can still be a good choice for strain differentiation. https://www.biotech-asia.org/vol16no3/a-new-pcr-based-species-genotyping-differentiation-approach-in-entamoeaba/ Amplicon; Contig; Nucmer; pecies differentiation; STR; Strain Differentiation