[Establishment of a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification assay].

OBJECTIVE

To establish a nucleic acid assay for detection of Echinococcus granulosus based on recombinase-aided isothermal amplification (RAA) assay.

METHODS

The 12S rRNA gene of E. granulosus was selected as the target gene, and the specific primers and fluorescent probes for RAA assay were designed, screened and synthesized to establish a fluorescent RAA assay for detection of E. granulosus. The sensitivity of the fluorescent RAA assay was evaluated using different copy numbers of target gene sequence-contained recombinant plasmids and various concentrations of E. granulosus genomic DNA as templates, and the specificity of the fluorescent RAA assay was evaluated using the genomic DNA from E. granulosus, E. multilocularis, Schistosoma japonicum, S. mansoni, Ancylostoma duodenale, Clonorchis sinensis, Taenia saginata, Spirometra mansoni and Taenia solium as templates.

RESULTS

A fluorescent RAA assay was successfully established for detection of E. granulosus, which achieved specific amplification of E. granulosus genomic DNA within 20 min at 39 ℃. The lowest detection limit of the fluorescent RAA assay was 10 copies/μL of recombinant plasmids and 0.1 ng/μL E. granulosus genomic DNA, which exhibited a high sensitivity, and the fluorescent RAA assay was all negative for the genomic DNA from E. multilocularis, S. japonicum, S. mansoni, A. duodenale, C. sinensis, T. saginata, Spirometra mansoni and T. solium, which exhibited a high specificity. In addition, this fluorescent RAA assay successfully detected genomic DNA from E. granulosus cysts.

CONCLUSIONS

A rapid, sensitive and specific fluorescent RAA assay is successfully established for nucleic acid detection of E. granulosus.