title: Molecular discrimination between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand.
abstract: To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease.
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku Ward, Tokyo 162-8640, 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: Yosuke
authors.initials: Y
authors.lastname: Kameoka
authors.forename: Masanori
authors.initials: M
authors.lastname: Kawanaka
mesh_terms: D000818:Animals; D006801:Humans; D009710:Nucleotide Mapping; D010237:Paragonimiasis / Q000175:diagnosis / Q000469:parasitology*; D010238:Paragonimus / Q000145:classification / Q000235:genetics*; D048492:Paragonimus westermani / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013045:Species Specificity; D013785:Thailand