环介导同温DNA扩增技术鉴定血吸虫感染性钉螺方法的建立

Establishment of loop mediated isothermal DNA amplification for identifying Oncomelania snails infected with Schistosoma japonicum

目的建立一种快速鉴定血吸虫感染性钉螺的环介导同温DNA扩增方法。方法选择一个高拷贝的日本血吸虫基因SjR2的部分DNA序列作为扩增靶位,根据环介导同温DNA扩增原理设计合成引物,建立扩增该片段的环介导同温DNA扩增方法。使用此方法与聚合酶链反应(PCR)方法同时检测血吸虫感染性钉螺与正常钉螺的DNA,观察其应用价值。结果日本血吸虫SjR2基因的839～1 138 bp区段被成功扩增和克隆。利用根据该片段DNA序列设计合成的环介导同温DNA扩增反应的引物,成功地建立了能扩增该片段的环介导同温DNA扩增方法,其敏感性高于PCR法,能检出1 pg的血吸虫DNA。用此法检测30只感染性钉螺,阳性率为93.33%,而PCR的阳性率83.33%,两者差异有统计学意义(P<0.01)。结论环介导同温DNA扩增检测血吸虫感染性钉螺具有较高的敏感性,是一种潜在有效的血吸虫感染性钉螺鉴定方法。

Objective To establish a method of loop mediated isothermal DNA amplification for identifying the Oncornelania snails infected with Schistosorna japonicurn rapidly. Methods A partial sequence of S. japonicum gene SiR2 with high copy was selected as amplified target gene, and the primers were designed and synthesized following the mechanism of loop mediated isothermal DNA amplification, and were used to construct a loop mediated isothermal DNA amplification method for amplifying target DNA fragment. The value of this method for detecting S. japonicum infected snails was evaluated by detecting the snails infected with S. japonicum and negative snails. The snail samples were also detected by polymerase chain reaction （PCR） in parallel as control. Results The target DNA fragment from 839 to 1 138 bp in SiR2 gene of S. japonicurn was amplified and cloned. And a loop mediated isothermal DNA amplification method for detecting this fragment was established successfully. This method could detect one picogram target DNA molecule; and its sensitivity was higher than that of PCR. The positive rate was 93.33% when the DNA samples of 30 infected snails were detected by this method, but the positive rate of PCR was only 83.33%. There was a significant difference between these two methods（P〈0. 01）. Conclusion The sensitivity of loop mediated isothermal DNA amplification method of detecting the S. japonicum infected snails was higher, which will be a potential method for identifying the snails infected by S. japonicum.