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Updated: Dec 8, 2025
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
[Establishment of the gene detection method of Schistosoma mansoni based on the recombinase-aided isothermal
1Key Laboratory of National Health Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi 214064, China.
Objective:
To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of Schistosoma mansoni.
Methods:
The 121 bp highly-repeated sequence of S. mansoni 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 S. mansoni genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of S. japonicum, S. haematobium, Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity.
Results:
A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of S. mansoni 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 S. mansoni genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from S. japonicum, S. haematobium, A. duodenale and C. sinensis.
Conclusions:
A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of S. mansoni.
