体外转录法制备南芥菜花叶病毒RNA标准品  

Preparation of Arabis mosaic virus RNA standards by in vitro transcription

作　　者：赵晓丽[1] 周琦[1] 孙宁[1,2] 邓丛良[1] 

机构地区：[1]北京出入境检验检疫局,北京100026 [2]东南大学

出　　处：《植物检疫》2014年第1期37-40,共4页Plant Quarantine

基　　金：公益性行业(质检)科研专项(201110035)

摘　　要：本研究根据南芥菜花叶病毒外壳蛋白基因保守序列,设计合成了1对引物,在上游引物的5′端引入T7启动子,采用RT-PCR方法获得了体外转录模板,并对转录产物RNA的浓度及OD值进行了测定。将制备的RNA标准品10倍梯度稀释后制作实时荧光定量RT-PCR标准曲线,并利用实时荧光定量RT-PCR对所获得的RNA标准品进行稳定性指标的检测。结果表明,该模板具有良好的线性关系,相关系数为0.9989。该模板稳定性好,-80℃保存30 d后无显著变化。A pair of primers for RT-PCR was designed based on the conserved nucleotide sequence of coat protein gene sequence of Arabis mosaic virus （ArMV）, and T7 promoter was added to the 5＇end of the forward primer. RT- PCR was used to get the template for transcription in vitro, and the concentration and the OD value of the RNA standards were determined. Series of the obtained RNA standards with 10-fold dilution were used to establish the standard curves for real-time RT-PCR. Stability of the RNA standards quantified was detected by real-time RT-PCR. The result indicated that, the Ct value of plotted standard curve showed good linearity to the logarithm of copy number of template, with a R2 value of 0.9989. After storing at -80℃ for 30 days, the template still worked well, showing an excellent repetitiveness of the template.

关 键 词：南芥菜花叶病毒 体外转录 实时RT-PCR RNA标准品 

分 类 号：S432.41[农业科学—植物病理学]