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

重组酶介导的华支睾吸虫特异性核酸等温扩增方法的建立及初步评价-江苏血防所

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[Abstract] Objective: To establish a recombinase-aided isothermal nucleic acid amplification method (RAA) suitable for the detection of Clonorchis sinensis. Methods: Based on the I8S rRNA gene sequence of Clonorchis sinensis, specific primers and probes were designed, synthesized, and screened to develop a rapid fluorescence RAA assay for detecting this parasite. The assay was evaluated for its detection sensitivity using recombinant plasmids containing DNA fragments with varying copy numbers as templates, as well as genomic DNA from Clonorchis sinensis at different concentrations. Additionally, specificity was assessed by testing against genomic DNA from Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale, and Schistosoma mansoni. Finally, the ability of the method to detect field samples was preliminarily evaluated by extracting DNA from fecal samples containing Clonorchis sinensis eggs and from freshwater fish muscle samples harboring metacercariae. Results: A fluorescence RAA assay for Clonorchis sinensis was successfully established, enabling specific amplification of the parasite’s DNA within 20 minutes at 39°C. Using recombinant plasmids with varying DNA copy numbers as templates, the assay achieved a detection limit as low as 10 copies/L. When tested with genomic DNA from Clonorchis sinensis at different concentrations, the assay could detect as little as 3.7 μL of template. Importantly, no cross-reactivity was observed when the assay was applied to DNA samples from Ascaris lumbricoides, Echinococcus granulosus, Schistosoma japonicum, Ancylostoma duodenale, and Schistosoma mansoni. Furthermore, the assay successfully detected Clonorchis sinensis in human and rat fecal samples, as well as in fish samples, demonstrating its robust capability for on-site sample analysis. Conclusion: A simple, rapid, sensitive, and specific RAA method has been successfully developed for the detection of Clonorchis sinensis, offering a reliable tool for clinical and epidemiological applications.

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重组酶介导的核酸等温扩增荧光法快速检测日本血吸虫感染性钉螺-江苏血防所

重组酶介导的蓝氏贾第鞭毛虫特异性等温核酸扩增方法的建立及评价-江苏血防所

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重组酶介导的核酸等温扩增荧光法快速检测日本血吸虫感染性钉螺-江苏血防所

Next page

重组酶介导的蓝氏贾第鞭毛虫特异性等温核酸扩增方法的建立及评价-江苏血防所