CAJ | 학술논문

目的：建立一种快速检测日本血吸虫感染性钉螺的方法。方法采用环介导等温扩增技术（LAMP ）以日本血吸虫的特异基因为靶序列，利用Primer Explorer V3软件设计扩增靶基因的4条LAMP引物，酚氯仿法提取感染性钉螺中的日本血吸虫DNA ，取适量模板DNA加入LAMP反应体系进行对日本血吸虫靶基因扩增，经肉眼观察、SYBR Green I染色电泳及测DNA序列来判定扩增产物。在99个阴性钉螺中各加入1个阳性钉螺，其中每个阳性钉螺分别感染日本血吸虫毛蚴数量分别为10、8、6、4、2、1条。结果感染性钉螺检测管经显色呈绿色为阳性，阴性钉螺呈棕色；日本血吸虫的特异基因经LAMP扩增后电泳呈现LAMP特征性梯状条带，阴性钉螺及感染肝吸虫不出现LAMP特征性梯状条带；扩增产物经酶切及序列分析显示为目的基因；在100个钉螺中只要有2条日本血吸虫毛蚴感染便可检出。结论环介导等温扩增技术（LAMP）可快速有效检测日本血吸虫感染性钉螺，有望为疫区日本血吸虫感染性钉螺高效快速检测提供一种新方法。
목적：건립일충쾌속검측일본혈흡충감염성정라적방법。방법채용배개도등온확증기술（LAMP ）이일본혈흡충적특이기인위파서렬，이용Primer Explorer V3연건설계확증파기인적4조LAMP인물，분록방법제취감염성정라중적일본혈흡충DNA ，취괄량모판DNA가입LAMP반응체계진행대일본혈흡충파기인확증，경육안관찰、SYBR Green I염색전영급측DNA서렬래판정확증산물。재99개음성정라중각가입1개양성정라，기중매개양성정라분별감염일본혈흡충모유수량분별위10、8、6、4、2、1조。결과감염성정라검측관경현색정록색위양성，음성정라정종색；일본혈흡충적특이기인경LAMP확증후전영정현LAMP특정성제상조대，음성정라급감염간흡충불출현LAMP특정성제상조대；확증산물경매절급서렬분석현시위목적기인；재100개정라중지요유2조일본혈흡충모유감염편가검출。결론배개도등온확증기술（LAMP）가쾌속유효검측일본혈흡충감염성정라，유망위역구일본혈흡충감염성정라고효쾌속검측제공일충신방법。
Objective To set up a method to detect schistosoma Japonicum -infected oncomelania hupensis straightly and promptly .Methods The loop-mediated isothermal amplification (LAMP ) was applied .Four primers were designed to target gene by Primer Explorer V 3 .The DNA of Schistosoma japonicum was extracted by phenol-chloroform extraction .LAMP results were judged by naked eye ,SYBR Green I stain and electrophoretic analys-is .Adding 1 Positive snails in 99 negative snails in which each positive snail is infected 10 to 1 schistosoma Ja-ponicum .Results Positive signal was observed with S .japonicum -infected snails tube ,whose color is green . While those in control groups are brown .We can observed characteristic DNA ladder in positive tube while we can’t find it in those negative one .The sequencing result of antisence fragments was in accordance with the known gene after digested by Alu I .There was 2 S .japonicum-infected snails among 100 negative control snails per re-action in the limit of detection for S .japonicum-infected snails .Conclusion It is obvious that the LAMP assay is a new ,prompt ,sensitive and straight method of detecting Schistosoma Japonicum -Infected Oncomelania hupensis Samples in the infected area the and it can be used for mass detection .