Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica, Entamoeba dispar and Entamoeba mosh¬kovskii

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. 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. 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. 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.