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Focusing on the research and development and application of isothermal nucleic acid amplification technology

基于重组酶介导核酸等温扩增反应的曼氏血吸虫基因检测方法的建立-江苏血防所

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Objective: To establish a recombinase-aided amplification (RAA) method using nucleic acid isothermal amplification, which can specifically detect the gene fragment unique to Schistosoma mansoni. Methods: A highly repetitive 121-bp gene fragment from Schistosoma mansoni was selected as the target sequence. Based on the principle of RAA reaction, primers and a fluorescent probe were designed and synthesized to develop and optimize the fluorescence-based RAA assay system. The sensitivity of this method was evaluated by performing fluorescence RAA amplification reactions with recombinant plasmids containing varying numbers of the 121-bp gene fragment, as well as with genomic DNA samples of Schistosoma mansoni at different concentrations. Additionally, the specificity of the assay was assessed by testing it against genomic DNA extracted from Japanese schistosome eggs, Egyptian schistosome eggs, duodenal hookworm eggs, and Clonorchis sinensis metacercariae. Results: The established fluorescence-based RAA method demonstrated specific amplification of Schistosoma mansoni genomic DNA within 20 minutes at 39°C. When using recombinant plasmids as templates, the method could detect as few as 10 plasmid copies per microliter. In contrast, when genomic DNA was used as the template, the assay achieved a detection limit as low as 0.1 femtogram per microliter. Importantly, no positive signals were observed when the assay was applied to genomic DNA derived from Japanese schistosome eggs, Egyptian schistosome eggs, duodenal hookworm eggs, or Clonorchis sinensis metacercariae. Conclusion: A fluorescence-based RAA method capable of detecting Schistosoma mansoni DNA has been successfully developed. This method is characterized by its rapidity, ease of operation, and excellent sensitivity and specificity, making it a promising tool for the diagnosis of Schistosoma mansoni infections.

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基于重组酶介导等温扩增技术的细粒棘球绦虫核酸检测方法的建立-江苏血防所

加强分子诊断方法的研发应用 助力我国精准血防-江苏血防所

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基于重组酶介导等温扩增技术的细粒棘球绦虫核酸检测方法的建立-江苏血防所

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加强分子诊断方法的研发应用 助力我国精准血防-江苏血防所