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利用改进的CTAB法提取出优质的草莓总RNA,可以稳定地进行RT PCR。针对草莓斑驳病毒(SMoV)基因组序列设计筛选引物,对病毒基因组的不同区域进行扩增,扩增产物经克隆、测序证明为SMoV的特异片段,与国外序列的同源性为91% ~97%。为了监测RNA的质量和RT PCR反应的正常进行,在检测体系中引入线粒体NADH脱氢酶基因ND2亚基作为内标,内标引物跨越内含子区域,只对剪接后的mRNA进行特异扩增,可以较好地监测整个检测过程。在国内首次建立了SMoV的RT PCR检测体系,以优质的草莓总RNA为模板,结合扩增内标的检测体系,对草莓病毒指示植物和草莓栽培品种都可以进行快速稳定的检测。
Using improved CTAB method to extract high quality strawberry total RNA, RT PCR can be stably carried out. According to the sequence of SMoV genome, primers were screened and the different regions of the virus genome were amplified. The amplified product was cloned and sequenced to be a specific fragment of SMoV. The homology was 91% -97% . In order to monitor the quality of RNA and the normal progress of RT PCR reaction, the ND2 subunit of mitochondrial NADH dehydrogenase gene was introduced into the detection system as an internal standard. The internal standard primers spanned the intron region and only spliced mRNA was specifically expanded Increased, you can better monitor the entire testing process. For the first time in China, the RT-PCR detection system of SMoV was established. With the high-quality strawberry total RNA as a template and the detection system of amplified internal standard, rapid and stable detection of strawberry virus-indicating plants and strawberry cultivars can be carried out.