title: Application of multiplex pcr for species discrimination using individual metacercariae of Paragonimus occurring in Thailand.
abstract: A total of 6 lung fluke species have been documented in Thailand, of which P. heterotremus is the most important, since it affects humans. Although P. westermani is found as metacercariae in the same crab species as P. heterotremus in Thailand, human infections with P. westermani have not been confirmed. To accurately discriminate between the individual metacercariae of these two species, we established a multiplex PCR method. Using this method, two products each were amplified from the metacercarial DNA samples of P. heterotremus (ca. 310 and 520 bp) and P. westermani (ca. 140 and 520 bp). In contrast, 520-bp products alone were found to be generated from the DNA samples of P. siamensis, P. bangkokensis and P. harinasutai, 3 other species of lung flukes known to occur in Thailand. Digestion of these 520-bp products with the restriction enzyme ScrFI could unequivocally discriminate species by the number and size of the produced band(s): 3 bands (ca. 60, 210 and 250 bp) for P. harinasutai, 2 bands (ca. 250 and 270 bp) for P. bangkokensis, and an uncut band (520 bp) for P. siamensis. The established multiplex PCR used in combination with restriction enzyme digestion (PCR-RFLP with ScrFI) is effective for discriminating the 5 different species of lung flukes occurring in Thailand, even at the metacercarial stage.
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan. hsugi@nih.go.jp
authors.forename: Hiromu
authors.initials: H
authors.lastname: Sugiyama
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Morishima
authors.forename: Achariya
authors.initials: A
authors.lastname: Rangsiruji
authors.forename: Sutheewan
authors.initials: S
authors.lastname: Binchai
authors.forename: Punsin
authors.initials: P
authors.lastname: Ketudat
authors.forename: Masanori
authors.initials: M
authors.lastname: Kawanaka
mesh_terms: D000818:Animals; D003386:Brachyura / Q000469:parasitology; D018509:DNA, Helminth / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005819:Genetic Markers; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D010238:Paragonimus / Q000145:classification* / Q000235:genetics; D010784:Photomicrography; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013045:Species Specificity; D013785:Thailand
chemical_list: D018509:DNA, Helminth; D005819:Genetic Markers