Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes
of diarrheal and intestinal diseases all over the world. Microscopic methods are useful
in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed
approximately 60 percent. Recently, molecular techniques have been used increasingly
for the identification and characterization of the parasites. Among those, in this
study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA)
for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii,
G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from
12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive
for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the
samples, multiplex PCR was done for detection and differentiation of above mentioned
parasites. QPCR-MCA was also performed for the detection and differentiation of 11
isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex
PCR was able to simultaneous detect and differentiate of above mentioned parasite
in a single reaction. QPCR-MCA was able to differentiate genus and species those five
protozoa using melting temperature simultaneously at the same time and temperature
programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation
of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium
spp. Application of multiplex PCR for detection of more than one species in a test
in developing countries, at least in reference laboratories has accurate diagnosis
and plays a critical role in differentiation of protozoan species. Multiplex PCR assay
with a template and multi template had different results and it seems that using a
set of primers with one template has higher diagnostic capability in compare with
multi template. The results of this study showed that the use of the qPCR-MCA can
be an effective method to simultaneous distinguish of the above mentioned parasites.