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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。
In this study, a pair of primers was designed and synthesized based on the conserved sequence of the coat protein gene of Arabidopsis mosaic virus. The T7 promoter was introduced at the 5 ’end of the upstream primer, and the transcription template was obtained by RT-PCR. Concentration and OD values were determined. Real-time fluorescence quantitative RT-PCR standard curve was prepared by 10-fold gradient dilution of the prepared RNA standard, and the stability of the obtained RNA standard was detected by real-time fluorescence quantitative RT-PCR. The results show that the template has a good linear relationship with a correlation coefficient of 0.9989. The template has good stability and no significant change after 30 d storage at -80 ℃.