title: Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method.
abstract: Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes.
authors.affiliation: Department of Medical Parasitology and Mycology, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Mohammad Bagher
authors.initials: MB
authors.lastname: Rokni
authors.forename: Hossein
authors.initials: H
authors.lastname: Mirhendi
authors.forename: Azadeh
authors.initials: A
authors.lastname: Mizani
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mohebali
authors.forename: Mitra
authors.initials: M
authors.lastname: Sharbatkhori
authors.forename: Eshrat Beigom
authors.initials: EB
authors.lastname: Kia
authors.forename: Hamid
authors.initials: H
authors.lastname: Abdoli
authors.forename: Shahrokh
authors.initials: S
authors.lastname: Izadi
mesh_terms: D000003:Abattoirs; D000818:Animals; D001483:Base Sequence; D002020:Buffaloes; D002417:Cattle; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D018509:DNA, Helminth / Q000737:chemistry / Q000302:isolation & purification*; D003937:Diagnosis, Differential; D004587:Electrophoresis, Agar Gel; D005209:Fasciola / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005210:Fasciola hepatica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005211:Fascioliasis / Q000175:diagnosis / Q000469:parasitology*; D006041:Goats; D007492:Iran; D008099:Liver / Q000469:parasitology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D016415:Sequence Alignment; D012756:Sheep
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D004262:DNA Restriction Enzymes