Iranian Journal of Parasitology 2014. 9(4):466-473.

Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii.
Nozhat Zebardast, Ali Haghighi, Farshid Yeganeh, Seyyed Javad Seyyed Tabaei, Mohammad Javad Gharavi, Shirzad Fallahi, Zohreh Lasjerdi, Nima Salehi, Niloofar Taghipour, Cobra Kohansal, Farideh Naderi


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.


DNA extraction; Entamoeba dispar; Entamoeba hitolytica; Entamoeba moshkovskii; Multiplex PCR

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