[Establishment of the gene detection method of Schistosoma mansoni based on the recombinase-aided isothermal amplification assay].

To establish a recombinase-aided isothermal amplification (RAA) assay for nucleic acid detection of Schistosoma mansoni.The 121 bp highly-repeated sequence of S. mansoni was selected as the target gene fragment to be detected. The primers and fluorescent probes were designed using the Amplfix software, and a fluorescent RAA assay was established and optimized. The fluorescent RAA assay was performed to detect gradient diluent recombinant plasmids containing target gene fragment and different concentrations of S. mansoni genomic DNA to determine the sensitivity, and this assay was applied to detect the genomic DNA of S. japonicum, S. haematobium, Ancylostoma duodenale and Clonorchis sinensis to evaluate the specificity.A fluorescent RAA assay was successfully established, which was effective to amplify the specific gene fragments of S. mansoni within 20 min at 39 ℃. The minimum detectable limit of the fluorescent RAA assay was 10 copies/μL using recombinant plasmids as templates and 0.1 fg/μL using S. mansoni genomic DNA samples as templates. The fluorescent RAA assays were all negative for detecting the genomic DNA from S. japonicum, S. haematobium, A. duodenale and C. sinensis.A novel fluorescent RAA assay is successfully established, which is simple, rapid, sensitive and specific to detect genomic DNA of S. mansoni.［摘要］目的 建立一种可用于曼氏血吸虫特异性基因片段检测的重组酶介导的核酸等温扩增方法 (Recombinase-aid- ed amplification, RAA)。方法 以曼氏血吸虫121 bp高重复基因片段作为靶序列, 根据RAA反应原理设计、合成引物及 荧光探针, 建立并优化荧光RAA法反应体系。分别以不同拷贝数的含121 bp基因片段的重组质粒及不同浓度曼氏血吸 虫基因组DNA为模板进行荧光RAA法扩增, 评价该方法的敏感性; 分别以日本和埃及血吸虫虫卵、十二指肠钩虫虫卵、华支睾吸虫囊蚴基因组DNA为模板进行荧光RAA法检测, 评价其特异性。结果 建立的荧光RAA法可在39 ℃、20 min 内特异性扩增曼氏血吸虫基因组DNA。以重组质粒为模板, 荧光RAA法最低可检出的质粒拷贝数为10拷贝/μL; 以基 因组DNA为模板, 荧光RAA法最低可检测浓度为0.1 fg/μL。以日本血吸虫虫卵、埃及血吸虫虫卵、十二指肠钩虫虫卵、华支睾吸虫囊蚴基因组DNA为模板进行荧光RAA法检测, 结果均为阴性。结论 成功建立了一种可用于曼氏血吸虫 DNA检测的荧光RAA法, 该方法反应快捷、操作简便, 敏感性和特异性均较好。.