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Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development of a Recombinase-Aided Isothermal Amplification Assay for the Detection of Schistosoma japonicum-Specific Gene Fragments—Jiangsu Institute of Parasitic Diseases
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[Abstract] Objective: To establish a recombinase-mediated isothermal amplification (RAA) method that can be used for the detection of species-specific gene fragments of Schistosoma japonicum. Methods: A target sequence was selected from the SiG28 gene fragment of Schistosoma japonicum. Primers were designed and synthesized based on the principle of RAA reaction, and an optimized RAA reaction system was established and validated. The sensitivity of the RAA method was evaluated by simultaneously amplifying, using this method and polymerase chain reaction (PCR), TA-cloned plasmids containing SjG28 gene fragments at different copy numbers in gradient dilutions, as well as genomic DNA samples at varying concentrations. The specificity of the RAA method was assessed by testing genomic DNA from Schistosoma mansoni, Ascaris lumbricoides, and Ancylostoma duodenale, as well as genomic DNA from healthy human blood samples. Results: The established RAA method could specifically amplify genomic DNA from adult worms and eggs of the Chinese mainland strain of Schistosoma japonicum, and the reaction could be completed within 30 minutes. Using recombinant plasmids as templates, the RAA method could detect as few as 20 plasmid copies per microliter; when using genomic DNA as a template, the method could detect down to a concentration of 0.01 ng/μL. Amplification results obtained with the established RAA method using genomic DNA from whole blood of healthy individuals, as well as genomic DNA from adult Schistosoma mansoni, Ascaris lumbricoides, and Ancylostoma duodenale, were all negative. Conclusion: This study has developed an RAA method that is rapid, highly sensitive, and specific, and it holds great potential for application in the genetic diagnosis of schistosomiasis japonica.
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