Establishment and preliminary evaluation of recombinase aided isothermal amplification (RAA) assay for specific nucleic acid detection of Clonorchis sinensis / 中国血吸虫病防治杂志

ABSTRACT

Objective To establish a recombinase aided isothermal amplification (RAA) assay for detection of Clonorchis sinensis. Methods The 18S ribosomal RNA (18S rRNA) sequence of C. sinensis was used as the target sequence, and specific primers and probes were designed, synthesized and screened to establish a rapid fluorescent RAA assay for the detection of C. sinensis. Then, the sensitivity of the fluorescent RAA assay was evaluated using the recombinant plasmids containing various copy numbers of DNA fragments and C. sinensis genomic DNA at various concentrations, and the specificity of the fluorescent RAA as say was evaluated using the genomic DNA of Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale and S. mansoni as templates. DNA samples were extracted from the feces containing C. sinensis eggs and freshwater fish containing metacercaria for the fluorescent RAA assay, and the performance for detection of C. sinensis-infected samples was preliminarily assessed in the field. Results A fluorescent RAA assay for detection of C. sinensis was successfully established, which was feasible for specific amplification of C. sinensis genomic DNA at 39 °C within 20 min. The lowest detection limit was 10 copies/μL if the recombinant plasmid containing various copy numbers of DNA fragments was used as a template, and the lowest detection limit was 3 pg/μL if the C. sinensis genomic DNA at various concentrations served as a template. All detections were negative if the genomic DNA of A. lumbricoides, E. granulosus, S. japonicum, A. duodenale, and S. mansoni was used as templates. In addition, the fluorescent RAA assay showed a high performance for the detection of C. sinensis-infected samples in the field, which successfully detected C. sinensis-infected human and rat fecal samples and Pseudorasbora parva samples. Conclusion A fluorescent RAA assay is successfully established, which is simple, rapid, sensitivity and specific for detection of C. sinensis.