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

重组酶介导的日本血吸虫特异性基因片段核酸等温扩增检测方法的建立-江苏血防所

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[Abstract] Objective: To establish a recombinase-aided amplification (RAA) method suitable for detecting specific gene fragments of Schistosoma japonicum. Methods: A target sequence derived from the SiG28 gene of Schistosoma japonicum was used to design and synthesize primers based on the RAA reaction principle. An optimized RAA reaction system was then established and validated. The sensitivity of the method was evaluated by simultaneously amplifying gradient-diluted TA-cloned plasmids containing SjG28 gene fragments with varying copy numbers, as well as genomic DNA samples at different concentrations, using both RAA and polymerase chain reaction (PCR). Additionally, the specificity of the assay was assessed by testing genomic DNA from Schistosoma mansoni, Ascaris lumbricoides, and Ancylostoma duodenale, along with healthy human genomic DNA. Results: The established RAA method demonstrated specific amplification of genomic DNA from adult worms and eggs of the Chinese mainland strain of Schistosoma japonicum, with the entire reaction completed within 30 minutes. When using recombinant plasmid as the template, the lowest detectable plasmid copy number was 20 copies/μL; when genomic DNA was used as the template, the method could detect as low as 0.01 ng/μL. Furthermore, no amplification was observed when the assay was performed using healthy human whole-blood genomic DNA or genomic DNA from Schistosoma mansoni adults, Ascaris lumbricoides, and Ancylostoma duodenale as templates. Conclusion: This study successfully developed an RAA method that is rapid, highly sensitive, and specific, making it a promising tool for the genetic diagnosis of schistosomiasis japonica.

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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

重组酶介导的斯氏并殖吸虫等温扩增荧光检测方法的建立及检测效果初步评价

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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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重组酶介导的斯氏并殖吸虫等温扩增荧光检测方法的建立及检测效果初步评价