利用内标为基础的RT-PCR技术检测葡萄茎痘伴随病毒

 选取经RT-PCR检测确认带葡萄茎痘伴随病毒的‘全球红’葡萄品种。以葡萄叶片、韧皮部作为试材, 采用SDS法提取高质量总RNA作为模板, 以GRSPaV的特异互补引物引导, 反转录合成cDNA,通过PCR扩增, 获得830 bp的预期目的片段。以线粒体nad5基因为内标, 建立了GRSPaV与nad5共扩增体系, 将扩增的GRSPaV特异性片段及nad5目的片段分别克隆测序, 与NCB I中所提交的核酸序列进行比对, GRSPaV与序列号AF057136的同源性达96%; nad5与序列号D37958的同源性达96.67%。

Using RT-PCR Assay to Detect Grapevine rupestris stem pitting associated virus Based on Internal Control

This study prefer to establish a rapid, sensitive, accrual and practical detection system for Grapevine rupestris stem pitting associated virus (GRSPaV) based on internal control. These grapevine varieties such as‘Red Golbe’ that were detected to carry GRSPaV by RT-PCR were selected as testing material. Using SDS method to extract high quality total RNA from grape leaves and phloem, which was template to synthesize the cDNA by the guide of GRSPaV special antisense primer. After that, through PCR amplification, the 830 bp special fragmentwas gained. The co-amplification system of GRSPaV and plant mitochondrial nad5 as internal controlwas established. The use of internal control minimizes the risk of obtaining false negative RT-PCR results and avoids the need to eliminate contaminating DNA in extracts. The GRSPaV special fragment and nad5 aim fragmentwere cloned and sequenced respectively. The sequenced results aligned with nucleotide array in NCBI. The aligned results showed that GRSPaV has 96% similarity with array codeAF057136 and nad5 has 96.67% similarity with array code D37958.