体外转录法制备西尼罗河热病毒RNA片段  

The Preparation of West Nile Virus RNA Segment by In Vitro Transcription

作　　者：邹艳丽[1] 赵永刚[1] 张永强[1] 包静月[1] 孙成友[1] 王君玮[1] 李金明[1] 王志亮[1] 

机构地区：[1]中国动物卫生与流行病学中心国家外来动物疫病诊断中心,山东青岛266032

出　　处：《中国动物检疫》2011年第12期29-31,共3页China Animal Health Inspection

基　　金：十一五国家科技支撑计划(2006BAD06A13);青岛市科技发展计划项目(07-2-3-5-jch)

摘　　要：目的通过体外转录获得西尼罗河热病毒保守区基因的RNA片段,为核酸快速检测方法的建立和改进提供阳性定量标准品。方法设计西尼罗河热病毒基因保守区克隆引物,PCR从合成的基因DNA获得相应片段,连接至质粒PGEM-T-easy上并筛选阳性重组质粒,测序鉴定后酶切线性化,用T7RNA聚合酶进行体外转录,得到固定长度的RNA片段,DNase酶处理后测定浓度,梯度稀释后用RT-PCR验证。结果获得含西尼罗河热病毒靶基因序列的准确定量拷贝数的RNA片段,质量浓度分别为352.6ng/μL,RT-PCR验证均扩增出相应目的条带。结论获得的RNA片段可作为西尼罗河热病毒核酸快速检测方法的阳性定量标准品。Qbjective To prepare the positive standard substance containing the specific gene of West Nile virus （WNV） by in vitro transcription for the development and further improvement of the nucleic acid detection method of WNV. Methods Primers targeting the conservative segment of WNV were designed. The target gene was amplified by PCR and connected into the pGEM-T-easy vector. The plasmids were sequenced and the correct positive plasmid was selected then cutted into lined plasmid by restriction enzyme used as in vitro transcription template. After transcribed by T7 RNA polymerase and treated by DNase I, the fixed length RNA was generated. The concentration of RNA was assayed. The RNA was diluted and verified by RT-PCR. Results The RNA fragment containing WNV gene was obtained. The detection limit of RT-PCR was 5.0 × 10^2copies/μt L. Conclusion The RNA transcribed in vitro could be used as positive control in the detection of West Nile virus.

关 键 词：西尼罗河热病毒 T7启动子 体外转录 

分 类 号：S852.659.6[农业科学—基础兽医学]