摘要
目的通过体外转录获得登革热1-4型病毒保守区基因的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。方法设计登革热1-4型病毒基因保守区克隆引物,RT-PCR从病毒RNA获得相应片段,分别连接至质粒PGEM-T-easy上并筛选阳性重组质粒,测序鉴定后酶切线性化,用T7 RNA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。结果获得含登革热1-4型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352.6 ng/μl、384.2 ng/μl、457.3 ng/μl、368.7 ng/μl,RT-PCR验证均扩增出相应目的条带。结论获得的RNA片段可作为登革热1-4型病毒核酸快速检测方法的阳性定量标准品。
Objective To transcript RNA fragment of dengue virus1-4 type in vitro in order to obtain quantitative standard for detecting mRNA of dengue viruses.Methods According to the specific sequence of dengue viruses mRNA,the primers were designed and synthesized.The fragments generated by RT-PCR were cloned into the PGEM-T-easy vector with TA cloning protocol.The positive recombinant linearized plasmids were used to transcript RNA in vitro and the RNA were used as standard quantitative templates of RT-PCR method.Results Specific fragments were amplified from virus culture medium,which were confirmed by DNA cloning and sequencing,their concentrations were 352.6ng/μl,384.2ng/μl,457.3ng/μlm,and 368.7ng/μl respectively.cRNA of dengue in conserved region with precise quantification copy number were obtained.Conclusion Dengue viruses cRNA standards which could be used as quantitative standard for rapid detection method of nucleic acid of dengue viruses were prepared.
出处
《解放军预防医学杂志》
CAS
2010年第2期95-98,共4页
Journal of Preventive Medicine of Chinese People's Liberation Army
基金
南京军区重点课题(No.07Z043)
南京军区面上课题(No.08MB149)
关键词
登革热病毒
克隆
T7启动子
体外转录
dengue viruses
cloning
T7 Promoter
transcription in vitro
