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

重组酶介导的隐孢子虫属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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[Abstract] Objective: To establish a recombinase-aided amplification (RAA) method using recombinase-mediated isothermal nucleic acid amplification, which can be applied for Cryptosporidium detection, and to evaluate its sensitivity and specificity. Methods: A specific I8S rRNA gene sequence unique to the genus Cryptosporidium was selected as the target for detection. Primers and a fluorescent detection probe were designed and synthesized using Amplx software. An optimized RAA fluorescence reaction system was then established and validated. The sensitivity of the assay was assessed by using recombinant plasmids containing varying copy numbers of the 18S rRNA target sequence, as well as genomic DNA from Cryptosporidium oocysts at different concentrations, and pure Cryptosporidium oocyst DNA samples of varying quantities as templates. Specificity was evaluated by testing the assay with genomic DNA extracted from Cryptosporidium oocysts, Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella, and Shigella. Results: A reliable RAA-based method for detecting Cryptosporidium was successfully developed. This method enables efficient amplification of the Cryptosporidium genus-specific I8S rRNA gene fragment within 20 minutes at 39°C. Using recombinant plasmids with different copy numbers of the I8S rRNA target sequence, genomic DNA from Cryptosporidium oocysts at varying concentrations, and pure Cryptosporidium oocyst DNA samples in different quantities as templates, the assay demonstrated detection limits of 10 copies/μL, 1 ppb, and 1 oocyst/50 μL, respectively. Notably, no fluorescence signals were observed when genomic DNA from Giardia lamblia cysts, Schistosoma japonicum eggs, Ascaris lumbricoides eggs, Clonorchis sinensis eggs, Salmonella, or Shigella was used as the template. Conclusion: A robust RAA method suitable for detecting Cryptosporidium oocyst DNA has been successfully established. This method is simple to perform, offers rapid results, and exhibits excellent sensitivity and specificity.

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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

重组酶介导等温核酸扩增技术检测细棘球绦虫方法的建立及初步应用评价_周鸿让CDC

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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

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重组酶介导等温核酸扩增技术检测细棘球绦虫方法的建立及初步应用评价_周鸿让CDC