title: Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA.
abstract: 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.
authors.affiliation: Cellular and Molecular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran. Electronic address: Nszebardast@gmail.com.
authors.forename: Nozhat
authors.initials: N
authors.lastname: Zebardast
authors.affiliation: Department of Immunology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Farshid
authors.initials: F
authors.lastname: Yeganeh
authors.affiliation: Department of Parasitology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad Javad
authors.initials: MJ
authors.lastname: Gharavi
authors.affiliation: Department of Community and Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Alireza
authors.initials: A
authors.lastname: Abadi
authors.affiliation: Deptartment of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Seyyed Javad
authors.initials: SJ
authors.lastname: Seyyed Tabaei
authors.affiliation: Deptartment of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Electronic address: Ahaghighi11@yahoo.com.
authors.forename: Ali
authors.initials: A
authors.lastname: Haghighi
mesh_terms: D003458:Cryptosporidium / Q000302:isolation & purification*; D017931:DNA Primers; D004747:Entamoeba / Q000302:isolation & purification*; D004748:Entamoeba histolytica / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology*; D016829:Giardia lamblia / Q000302:isolation & purification*; D006801:Humans; D007411:Intestinal Diseases, Parasitic / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: Cryptosporidium spp.; Entamoeba spp.; Giardia lamblia; Multiplex PCR; QPCR-MCA; Simultaneous detection and differentiation