title: Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis.
abstract: We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.
authors.affiliation: Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Oranuch
authors.initials: O
authors.lastname: Sanpool
authors.forename: Pewpan M
authors.initials: PM
authors.lastname: Intapan
authors.forename: Tongjit
authors.initials: T
authors.lastname: Thanchomnang
authors.forename: Penchom
authors.initials: P
authors.lastname: Janwan
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
authors.forename: Pham Ngoc
authors.initials: PN
authors.lastname: Doanh
authors.forename: Hoang
authors.initials: H
authors.lastname: Van Hien
authors.forename: Do Trung
authors.initials: do T
authors.lastname: Dung
authors.forename: Wanchai
authors.initials: W
authors.lastname: Maleewong
authors.forename: Yukifumi
authors.initials: Y
authors.lastname: Nawa
mesh_terms: D000328:Adult; D000818:Animals; D003003:Clonorchiasis / Q000175:diagnosis* / Q000469:parasitology; D003004:Clonorchis sinensis / Q000145:classification / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000469:parasitology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D025202:Molecular Diagnostic Techniques; D009245:NADH Dehydrogenase / Q000235:genetics; D009889:Opisthorchiasis / Q000175:diagnosis* / Q000469:parasitology; D009891:Opisthorchis / Q000145:classification / Q000302:isolation & purification*; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D009245:NADH Dehydrogenase