Establishment of Duplex PCR for Identifying Metagonimus yokogawai and Haplorchis taichui

To develop a duplex PCR method for identifying Metagonimus yokogawai and Haplorchis taichui.ITS1 sequences of M. yokogawai and H. taichui, as well as those of their homologous species were obtained from GenBank, and two sets of specific primer pairs for M. yokogawai and H. taichui were designed accordingly using Primer Premier 5.0 software. PCR reaction system and conditions were optimized. The established duplex PCR method was applied in a pool of M. yokogawai, H. taichui, and 17 related species to examine its specificity. Sensitivity was evaluated through serial dilutions of plasmids containing their specific sequences. Finally, the duplex PCR was applied to identify M. yokogawai and H. taichui among trematodes collected from the viscera of 47 cats and 40 dogs to test its practicality.The duplex PCR method amplified target sequences of M. yokogawai and H. taichui, generating 648 bp and 279 bp products, respectively. No cross reaction was found with the following 17 related species: Haplorchis pumilio, Clonorchis sinensis, Pharyngostomum cordatum, the metacercaria of Metorchis sp. and Exorchis sp., Echinochasmus liliputanus, Echinochasmus perfoliatus, Echinostoma friedi, Hypoderaeum conoideum, Holostephanus sp., Diplodiscus sp., Anisakis sp., Metorchis orientais, Paragonimus westermani, Watsonius watsoni, Notocotylus sp. and Hysterothylacium sp, indicating a high specificity of this method. The detection limits for DNAs of M. yokogawai and H. taichui were 1.49 x 10(-1) pg and 1.14 x 10(-1) pg, suggesting a good sensitivity for this method. Further, the duplex PCR successfully identified M. yokogawai and H. taichui from cat and dog viscera, with no cross amplification of other trematodes.The duplex PCR is effective in identifying Metagonimus yokogawai and Haplorchis taichui.