[Establishment and evaluation of the detection method of specific DNA fragment by recombinase aided isothermal amplification].

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

Methods: Based on -specific 18S rRNA selected as the target gene to be detected, and the primer sequences and fluorescent probes designed using the software Amplfix, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect 18S RNA target sequence-contained recombinant plasmids at various copies, genomic DNA of oocysts at various concentrations, and genomic DNA extracted from various numbers of oocysts to assess the sensitivity of the assay, and to detect genomic DNA extracted from oocysts, cysts, eggs, eggs, eggs, and to determine the specificity of the assay.

Results: A fluorescent RAA assay was successfully established, which was effective to amplify the specific 18S RNA gene fragments of within 20 min at 39 ℃. The lowest limits of the fluorescent RAA assay were 10 copies/μL for detection of 18S RNA target sequence-contained recombinant plasmids at various copies, 1 pg/μL for detection of genomic DNA of oocysts at various concentrations, and one oocyst/μL for detection of genomic DNA extracted from various numbers of oocysts, and the fluorescent RAA assay was all negative for detection of genomic DNA from cysts, eggs, eggs, eggs, and

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