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Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development of a Gene Detection Method for Schistosoma mansoni Based on Recombinase-Aided Isothermal Nucleic Acid Amplification—Jiangsu Institute of Parasitic Diseases
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Objective: To establish a recombinase-mediated isothermal amplification method (Recombinase-Aided Amplification, RAA) that can be used for the detection of species-specific gene fragments of Schistosoma mansoni. Methods: A 121-bp highly repetitive gene fragment from Schistosoma mansoni was selected as the target sequence. Primers and a fluorescent probe were designed and synthesized based on the principle of RAA reaction, and a fluorescent RAA reaction system was established and optimized. The sensitivity of the fluorescent RAA method was evaluated by using recombinant plasmids containing the 121-bp gene fragment at different copy numbers and genomic DNA of Schistosoma mansoni at varying concentrations as templates. The specificity of the method was assessed by performing fluorescent RAA assays using genomic DNA extracted from eggs of Japanese schistosome and Egyptian schistosome, Ascaris duodenalis eggs, and Clonorchis sinensis metacercariae as templates. Results: The established fluorescent RAA method can specifically amplify genomic DNA of Schistosoma mansoni within 20 minutes at 39°C. When using recombinant plasmids as templates, the lowest detectable plasmid copy number by the fluorescent RAA method was 10 copies/μL; when using genomic DNA as templates, the lowest detectable concentration was 0.1 fg/μL. No positive results were obtained when the fluorescent RAA assay was performed using genomic DNA extracted from eggs of Japanese schistosome, Egyptian schistosome, Ascaris duodenalis eggs, and Clonorchis sinensis metacercariae as templates. Conclusion: A fluorescent RAA method suitable for the detection of Schistosoma mansoni DNA has been successfully established. This method features rapid reaction, simple operation, and good sensitivity and specificity.
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