title: Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.
abstract: Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Phiaw Chong
authors.initials: PC
authors.lastname: Foo
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Yean Yean
authors.initials: YY
authors.lastname: Chan
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Wei Cun
authors.initials: WC
authors.lastname: See Too
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Zi Ning
authors.initials: ZN
authors.lastname: Tan
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Weng Kin
authors.initials: WK
authors.lastname: Wong
authors.affiliation: Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia.
authors.forename: Pattabhiraman
authors.initials: P
authors.lastname: Lalitha
authors.affiliation: School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
authors.forename: Boon Huat
authors.initials: BH
authors.lastname: Lim
mesh_terms: D000818:Animals; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004747:Entamoeba / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004748:Entamoeba histolytica / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology; D005243:Feces / Q000469:parasitology*; D005612:Freeze Drying; D006801:Humans; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan