title: Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.
abstract: Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.
authors.affiliation: Department of Parasitology, National Center for Epidemiology, Budapest, Hungary.
authors.forename: Erika
authors.initials: E
authors.lastname: Orosz
authors.forename: Katalin
authors.initials: K
authors.lastname: Perkátai
authors.forename: Beatrix
authors.initials: B
authors.lastname: Kapusinszky
authors.forename: Agnes
authors.initials: A
authors.lastname: Farkas
authors.forename: István
authors.initials: I
authors.lastname: Kucsera
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004748:Entamoeba histolytica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004749:Entamoebiasis / Q000175:diagnosis* / Q000469:parasitology; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000469:parasitology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods; D016053:RNA, Protozoan / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013997:Time Factors
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D016053:RNA, Protozoan; D012336:RNA, Ribosomal, 16S