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Amoebiasis, a disease caused by  Entamoeba histolytica , is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the  E. histolytica / E. dispar  complex. Furthermore, morphologically similar species such as  Entamoeba hartmanni  contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for  E. histolytica  and  E. dispar  and a single real-time PCR for  E. hartmanni . The multiplex protocol detected up to 0.0143 pg of  E. histolytica  DNA and 0.5156 pg of  E. dispar  DNA, and the average melting temperature ( T    m  ) was 73°C and 70°C, respectively. For  E. hartmanni , the  T    m   was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested,  E. dispar  DNA was detected in 37; none exhibited  E. histolytica  DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however,  E. hartmanni  DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

Differential Diagnosis ofEntamoebaspp. in Clinical Stool Samples Using SYBR Green Real-Time Polymerase Chain Reaction