Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 9 4 2014 12 15 466 473 EN Nozhat Zebardast Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Ali Haghighi Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Farshid Yeganeh Dept. of Immunology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Seyyed Javad Seyyed Tabaei Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Mohammad Javad Gharavi Dept. of Parasitology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran. Shirzad Fallahi Dept. of Parasitology and Mycology, Lorestan University of Medical Sciences, Khorramabad, Iran. Zohreh Lasjerdi Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Nima Salehi Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Niloofar Taghipour Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cobra Kohansal Tamin Ejtemaee Hospital, Ahvaz, Iran. Farideh Naderi Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2015 10 14 Background: Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction (Multiplex PCR) is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. Methods: For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. Results: A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction. Conclusion: We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/376 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/376/368