利用内标为基础的RT-PCR技术检测草莓斑驳病毒

RT-PCR detection of Strawberry mottle virus based on internal control

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.

Highly pure total RNA for reverse transcription-polymerase chain reaction (RT-PCR) was extracted from strawberry with modified CTAB method. The specific segment of SMoV was amplified with the designed primers,and testified by cloning and sequencing. The results showed it shared 91%-97% nucleotide acid identity with the published sequences. In order to detect the effectiveness of the RNA extraction and RT-PCR,the gene encoding mitochondrial NADH dehydrogenase ND2 subunit (ndh B gene) was used as an internal control. By locating one of the primers across the splice junction with the last two 3′ nucleotides being in the second exon,the primers were shown to amplify only the spliced RNA derived cDNA but not the intron containing DNA. This is the first report that SMoV was detected by RT-PCR in China. By using total RNA as template,in combination with specific internal control,the detection provides a quick and effective method for routine diagnosis of SMoV in strawberry cultivars and virus indicator plants.