Objective: To establish a novel method for the detection of Schistosoma japonicum specific gene fragments by recombinase aided isothermal amplification (RAA).
Methods: The gene fragment SjG28 of S. japonicum was selected as the target gene fragment to be detected, and the primers were designed according to the mechanism of RAA reaction. The reaction of isothermal amplification of S. japonicum was established and optimized. Then this method was applied to amplify and detect the specific gene fragment in the gradient diluent SjG28-recombiant plasmids and different concentrations of S. japonicum genomic DNA to estimate the sensitivity of this method. The samples were also detected by polymerase chain reaction (PCR) in parallel as control. This method was applied to detect the genomic DNA of S. mansoni, Ascaris lumbricoides, and Ancylostoma duodenale to evaluate the specificity.
Results: The specific gene fragment was amplified from genomic DNA of adult worms and eggs of S. japonicum by recombinase aided isothermal amplification reaction established in this study. The reaction can be completed within 30 minutes and the minimum detectable template was 20 copies of plasmids or 0.5 ng of genomic DNA per microliter. Other parasites'genomic DNAs, such as S. mansoni, A. lumbricoides, An. duodenale and healthy human blood genomic DNA were not able to be detected by this method.
Conclusions: A novel method for the detection of S. japonicum specific gene fragments by recombinase aided isothermal amplification is established in this study, which can be carried out conveniently and rapidly with a considerable sensitivity and specificity, showing the prospect for application in the diagnosis of schistosomiasis japonica.
[摘要] 目的 建立一种可用于日本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 (RAA) 。方法 以 日本血吸虫SjG28基因片段作为靶序列, 根据RAA反应原理设计合成引物, 建立并优化RAA反应体系。应用此方法与 聚合酶链式反应 (PCR) 同时扩增梯度稀释的含不同拷贝数的SjG28基因片段TA克隆质粒及不同浓度的基因组DNA, 以 评价其敏感性; 应用此方法检测曼氏血吸虫、似蚓蛔线虫、十二指肠钩口线虫基因组DNA及健康人血基因组DNA, 以评 价其特异性。结果 建立的RAA法可特异性扩增日本血吸虫中国大陆株成虫及虫卵基因组DNA, 反应可在30 min内完 成。以重组质粒为模板, RAA法最低可检出的质粒拷贝数为20个/μL; 以基因组DNA为模板, RAA法最低可检测浓度为 0.01 ng/μL。建立的RAA法以健康人全血基因组DNA及曼氏血吸虫成虫、似蚓蛔线虫、十二指肠钩口线虫基因组DNA 为模板的扩增结果均为阴性。结论 本研究建立了一种反应快捷、敏感性和特异性均较高的RAA方法, 其具有应用于 日本血吸虫病基因诊断的价值。.
Keywords: Gene fragment; Isothermal amplification of nucleic acid; Recombinase; Schistosoma japonicum.