title: A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs in faeces.
abstract: BACKGROUND
  Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method.
  
  
  METHODS
  In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs.
  
  
  RESULTS
  This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99±0.08, 62.12±0.15 and 74.10±0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tm values. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites.
  
  
  CONCLUSIONS
  Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections.
authors.affiliation: Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand Center of Excellence in Stem Cell Research, Thammasat University, Pathumthani, Thailand Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Chairat
authors.initials: C
authors.lastname: Tantrawatpan
authors.affiliation: Walai Rukhavej Botanical Research Institute (WRBRI), Mahasarakham University, Maha Sarakham, Thailand weerachai.s@msu.ac.th.
authors.forename: Weerachai
authors.initials: W
authors.lastname: Saijuntha
authors.affiliation: Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand Center of Excellence in Stem Cell Research, Thammasat University, Pathumthani, Thailand.
authors.forename: Sirikul
authors.initials: S
authors.lastname: Manochantr
authors.affiliation: Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand Center of Excellence in Stem Cell Research, Thammasat University, Pathumthani, Thailand.
authors.forename: Pakpoom
authors.initials: P
authors.lastname: Kheolamai
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand Faculty of Medicine, Mahasarakham University, Mahasarakham, Thailand.
authors.forename: Tongjit
authors.initials: T
authors.lastname: Thanchomnang
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Lakkhana
authors.initials: L
authors.lastname: Sadaow
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Pewpan M
authors.initials: PM
authors.lastname: Intapan
authors.affiliation: Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Wanchai
authors.initials: W
authors.lastname: Maleewong
mesh_terms: D000818:Animals; D003937:Diagnosis, Differential; D004449:Echinostoma / Q000302:isolation & purification*; D004451:Echinostomiasis / Q000175:diagnosis*; D005209:Fasciola / Q000302:isolation & purification*; D005211:Fascioliasis / Q000175:diagnosis*; D005243:Feces / Q000469:parasitology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D010237:Paragonimiasis / Q000175:diagnosis*; D010238:Paragonimus / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
keywords: Detection; Echinostoma malayanum; Faeces; Fasciola gigantica; Paragonimus heterotremus; Real-time FRET PCR