目的 结合重组酶介导的核酸等温扩增（recombinase aided amplification,RAA）和荧光探针方法建立日本血吸虫特异性基因片段的快速检测方法。方法 以日本血吸虫G28（SjG28）基因片段作为靶序列,应用DNAMAN 7.0软件设计合成引物及荧光探针,建立荧光RAA反应体系。扩增不同拷贝数的SjG28重组质粒,评价荧光RAA检测的敏感性;分别以日本血吸虫、曼氏血吸虫、细粒棘球绦虫、十二指肠钩口线虫、似蚓蛔线虫、华支睾吸虫基因组为模板进行荧光RAA检测,以评价该方法的特异性。采用荧光RAA分别检测500、200和50条日本血吸虫尾蚴感染的兔粪中虫卵DNA。分别在50只阴性钉螺中混入1、2、3、4、5、10只阳性钉螺,并提取DNA进行荧光RAA检测。结果 以不同拷贝数的SjG28重组质粒为模板进行荧光RAA扩增,在5 min时即可观察到明显的荧光信号,随着拷贝数的不断降低,检测到荧光信号的时间也随之延长,不同拷贝数的扩增均在10 min内完成,最低可检出的质粒浓度为10拷贝/μl。以曼氏血吸虫、细粒棘球绦虫、十二指肠钩口线虫、似蚓蛔线虫及华支睾吸虫基因组DNA为模板的荧光RAA扩增结果均为阴性。不同数量尾蚴感染的兔粪中提取的DNA样品经荧光RAA检测,均能得到有效的扩增,检测可在15 min内完成。含不同数量阳性钉螺的50只阴性钉螺提取的日本血吸虫DNA经荧光RAA检测,均能得到有效的扩增,检测可在10 min内完成。结论 本研究建立的可检测日本血吸虫核酸片段的荧光RAA方法,反应快捷,敏感性和特异性均较高。

Objective To establish a novel and rapid method to detect Schistosoma japonicum specific gene fragment using a real-time recombinase aided isothermal amplification (RAA) combined with a fluorescent probe. Methods S. japonicum G28（SjG28） was selected as the target gene to be detected, and the primers and fluorescent probes were designed accordingly. A real-time fluorescent RAA using RAA combined with fluorescent probe was established to detect SjG28. The sensitivity and specificity of the real-time fluorescent RAA was determined and optimized by using gradient dilutions of SjG28 recombinant plasmid as template and the genomic DNAs from other helminths of S. mansoni, Echinococcus granulosus, Ancylostoma duodenale, Ascaris lumbricoides and Clonorchis sinensis as controls. Then this method was applied to detect SjG28 in genomic DNA extracted from snails and feces of rabbits infected with S. japonicum. Results As low as 10 copies of SjG28 plasmid DNA could be detected by the developed real-time fluorescent RAA within 10 min. This method was able to detect SjG28 in the fecal samples from rabbit infected with different dose of cercarias (20, 200 and 500) within 15 min and snail pool (50) mixed with 1, 2, 3, 4, 5 and 10 cercaria-positive snails within 10 min. There was no product amplified from genomic DNAs extracted from other helminths: S. mansoni, E. granulosus, A. duodenale, A. lumbricoides and C. sinensis. Conclusion A novel and rapid florescent real-time fluorescent RAA method was established to detect S. japonicum with high sensitivity and specificity.