[Establishment of the gene detection method of based on the recombinase-aided isothermal amplification assay].

OBJECTIVE

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

METHODS

The 121 bp highly-repeated sequence of was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of , , and to evaluate the specificity.

RESULTS

A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from , , and .

CONCLUSIONS

A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of .