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Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development of a Recombinase-Aided Isothermal Amplification Fluorescence Detection Method for Paragonimus skrjabini and Preliminary Evaluation of Its Detection Performance
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[Abstract] Objective: To establish a rapid nucleic acid detection method for Paragonimus skrjabini based on recombinase-aided isothermal amplification (RAA) technology and to preliminarily evaluate its detection performance. Methods: Paragonimus skrjabini, Paragonimus westermani, and Metagonimus yokogawai metacercariae were isolated from freshwater crabs, and their genomic DNA was extracted for molecular identification. Primers and probes targeting the cytochrome c oxidase 1 gene (cox1) of Paragonimus skrjabini were designed, prepared, and screened. The fluorescent RAA assay was validated using genomic DNA from Paragonimus skrjabini metacercariae collected in Jiyuan City and Yiyang County, Luoyang City, Henan Province, as templates. Fluorescent RAA amplification was performed using recombinant plasmids containing different concentrations of the Paragonimus skrjabini cox1 gene sequence and genomic DNA from Paragonimus skrjabini metacercariae as templates to evaluate the detection sensitivity. The established fluorescent RAA method was applied simultaneously to detect genomic DNA from Paragonimus westermani, Metagonimus yokogawai, Clonorchis sinensis, and Schistosoma japonicum to assess its specificity. Results: Paragonimus skrjabini, Paragonimus westermani, and Metagonimus yokogawai metacercariae were successfully isolated from freshwater crab samples and confirmed by molecular identification and phylogenetic analysis to be homologous to the reference strains of Paragonimus in GenBank. A fluorescent RAA assay for Paragonimus skrjabini was successfully established, enabling amplification of genomic DNA from Paragonimus skrjabini metacercariae collected in Jiyuan City and Yiyang County, Luoyang City, Henan Province within 5 minutes. No amplification was observed in the negative control. Using recombinant plasmids as templates, the limit of detection for the fluorescent RAA assay was 10 copies per liter, with positive amplification occurring within 5 minutes. When genomic DNA was used as the template, the lowest detectable DNA concentration was 10 pg/μL, and positive amplification was achieved within 10–15 minutes. The fluorescent RAA assay yielded negative results when applied to genomic DNA from Paragonimus westermani, Metagonimus yokogawai, Schistosoma japonicum, and Clonorchis sinensis.
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