【摘要】 目的建立一套系统、完整的粪便日本血吸虫虫卵DNA提取法及PCR优化体系。方法利用蛋白酶K和苯酚法从感染日本血吸虫尾蚴的家兔粪便中提取DNA,根据日本血吸虫基因(AF00369)设计特异性引物,以粪便中提取的DNA为模板,采用PCR方法扩增目的基因片段,对PCR方法的各种反应条件进行优化并采用有限稀释法推测PCR检测法可检测到的最低虫卵数。结果以感染家兔的粪便中提取的DNA为模板,用PCR方法可扩增到分子量大小约为400 bp的特异性条带,测序结果经BLAST工具进行分析,与日本血吸虫基因(AF00369)比对的同源性为96%;25μl PCR反应体系的最优化反应条件:MgCl2 1.5μl;dNTP 0.5μl;引物1.0μl;DNA模板5.0μl;Taq DNA聚合酶0.5μl。扩增程序为:94℃预变性5min、94℃变性30s、53℃退火30s、72℃延伸30s,30个循环、72℃延伸7min、4℃保存。PCR检测法可检测到的最低虫卵数为0.015个。结论成功建立感染家兔粪便中虫卵DNA的提取方法及PCR血吸虫基因片段检测方法,同时对PCR反应条件进行优化,为从分子角度检测日本血吸虫虫卵DNA提供科学的实验依据。更多还原

【Abstract】 Objective To develop the method to extract the S.japonicum eggs DNA in feces and optimize the PCR system.Methods Protein K and Phenol were used to extract DNAs from stool samples of rabbits infected with S.japonicum, specific primers were designed according to the gene sequences of S.japonicum(AF00369), then took the DNAs which extracted from feces as template, PCR amplification was performed in the target gene fragment.The reaction systems of PCR among different react conditions were optimized and the method of limited dilution to test the minimum eggs which can be detected was adopted.Results The experiments above mentioned by PCR assay showed that the specific band sized 402bp was obtained.The sequenced gene analyzed by BLAST tool showed that the ratio of homology compared with the genome of S.japonicum(AF00369) was 96%;The optimal react conditions of PCR system were determined as followed:10× PCR Buffer 2.5μl;MgCl2 1.5μl;dNTP 0.5μl;primer 1.0μl;template DNA 5.0μl and Taq DNA polymerase 0.5μl in total 25μl reaction volume.Modified thermal profile consisted of an initial denaturation step at 94℃ for 5 min, followed by 30 cycles of 94℃ for 30s, 53℃ for 30s and 72℃ for 30s, then exposure to 72℃ for 7min and keep in 4℃ for reservation.Experiments showed that the minimum eggs DNA being detected was 0.015.Conclusion The method to extract DNAs in feces infected with S.japonicum and detect gene product of S.japonicum by PCR had been successfully established, and the PCR system had been optimized, which provided scientific experimental basis on the molecular level for detecting eggs DNA of S.japonicum.更多还原