Innovative Technology

Innovation

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

结合重组酶介导的核酸等温扩增和荧光探针快速检测日本血吸虫基因片段-江苏血防所

  • File size: 458.2KB

[Abstract] Objective: To establish a rapid detection method for Schistosoma japonicum-specific gene fragments by combining recombinase-aided amplification (RAA) with a fluorescence probe approach. Methods: The G28 (SjG28) gene fragment of Schistosoma japonicum was used as the target sequence. Primers and a fluorescence probe were designed and synthesized using DNAMAN 7.0 software to develop a fluorescent RAA reaction system. The sensitivity of the fluorescent RAA assay was evaluated by amplifying sic28 recombinant plasmids at varying copy numbers. Specificity was assessed by performing fluorescent RAA tests using genomic DNA templates from Schistosoma japonicum, Schistosoma mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides, and Clonorchis sinensis. Additionally, fluorescent RAA was employed to detect DNA extracted from eggs in feces of mice infected with 500, 200, and 50 S. japonicum cercariae. Furthermore, DNA was extracted from 50 uninfected Oncomelania snails spiked with 1, 2, 3, 4, 5, or 10 positive snails, followed by fluorescent RAA analysis. Results: Fluorescent RAA amplification using SjG28 recombinant plasmids at different copy numbers yielded clear fluorescence signals detectable within 5 minutes, with the time required to observe fluorescence gradually increasing as plasmid copy numbers decreased. All amplifications, regardless of copy number, were completed within 10 minutes, with the lowest detectable plasmid concentration being 10 copies/μL. Negative results were obtained when genomic DNA from Schistosoma mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides, and Clonorchis sinensis served as templates. Moreover, DNA extracted from mouse feces containing varying doses of S. japonicum cercariae consistently produced robust amplification via fluorescent RAA, with detection completed within 15 minutes. Finally, DNA extracted from 50 uninfected Oncomelania snails spiked with different numbers of positive snails also yielded efficient amplification via fluorescent RAA, achieving detection within 10 minutes. Conclusion: The fluorescent RAA method established in this study offers a rapid, highly sensitive, and specific approach for detecting Schistosoma japonicum nucleic acid fragments.

Previous page

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

实时荧光重组酶介导核酸扩增在疟原虫快速检测中的应用研究-浙江国旅&丽水、萧山检验检疫

Next page

Previous page

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

Next page

实时荧光重组酶介导核酸扩增在疟原虫快速检测中的应用研究-浙江国旅&丽水、萧山检验检疫