Establishment of a fluorescent recombinase-aided isothermal amplification assay for nucleic acid detection of Paraginiumus skrjabini and preliminary evaluation of its detection efficiency

Abstract

To establish a nucleic acid assay for detection of Paragonimus skrjabini based on the recombinase-aided isothermal amplification (RAA) technique, and to preliminarily evaluate its detection efficiency. The metacercariae of P. skrjabini, P. westermani and Euparagonimus cenocopiosus were isolated from crabs, and genomic DNA was extracted for molecular characterization. The cytochrome coxidase 1 (cox1) gene sequence of P. skrjabini was selected as the target gene fragment, and the primers and probes were designed, screened and synthesized for RAA assay. The genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province were used as templates for verification of the fluorescent RAA assay. The fluorescent RAA assay was performed to detect different concentrations of plasmids containing target gene fragment and P. skrjabini metacercariae genomic DNA to determine the sensitivity. Fluorescent RAA assay was performed with recombinant plasmids containing P. skrjabini cox1 gene sequences at different concentrations and P. skrjabini genomic DNA as templates to evaluate its sensitivity, and the genomic DNA of P. westermani, E. cenocopiosus, Clonorchis sinensis and Schistosoma japonicum was detected with fluorescent RAA assay to evaluate its specificity. P. skrjabini, P. westermani and E. cenocopiosus metacercariae were isolated from crabs, respectively. Molecular characterization and phylogenetic analysis confirmed their homology with the genes sequences of standard Paragonimus strains in GenBank. A fluorescent RAA assay was successfully established for nucleic acid detection of P. skrjabini, and the genomic DNA of P. skrjabini metacercariae from Jiyuan City and Yiyang County of Luoyang City, Henan Province was amplified using the fluorescent RAA assay within 5 min, while the negative control was not amplified. If the recombinant plasmid containing P. skrjabini cox1 gene sequences was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 copies/μL, and positive amplification was observed within 5 min. If genomic DNA was used as templates, the fluorescent RAA assay showed the lowest detection limit of 10 pg/μL, and all positive amplifications were found within 5 to 10 min. In addition, the fluorescent RAA assay was tested negative for P. westermani, E. cenocopiosus, C. sinensis and S. japonicum. A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of P. skrjabini, which has potential values in rapid field detection and species identification in freshwater crabs in areas endemic for P. skrjabini.