RT-PCR检测甜菜坏死黄脉病毒及总RNA提取方法研究

The Studies on RT-PCR Detection Method of Beet Necrotic Yellow Vein Virus(BNYVV) and the Extraction Method of Total RNA

为选择较为适合甜菜总RNA的提取方法,构建和优化RT有效体系。以田间甜菜叶片、根毛及根表皮为材料,研究了提取甜菜总RNA的方法以及利用RT-PCR技术进行甜菜坏死黄脉病毒(BNYVV)的检测。研究表明,获得良好的RT-PCR扩增效果,RT体系中dNTPs浓度、引物、AMV、模板RNA的浓度要分别达到1 mmol/L,1μmol/L,0.1 U/μL,0.01μg/μL较好。PCR体系中dNTPs浓度、引物、TaqDNA聚合酶、Mg2+的浓度要分别达到0.1 mmol/L,0.1μmol/L,0.01U/μL,1.48 mmol/L效果较好。RT-PCR法对BNYVV检测,可作为甜菜品种(育种材料)早期抗丛根病性鉴定的依据之一。

Two methods of extraction of total RNA from sugarbeet leaves, cortexes and root hair were discussed. Optimized conditions for RT-PCR method for detecting BNYVV were also studied in this paper. The results were as follow, for getting the effective RT-PCR detection system, the better Concentration of RT reaction ingredients dNTPs, primer, AMV and template RNA were 1 mmol/L, 1 μmol/L, 0.1 U/μL, 0.01 μg/μL respectively. The concentration of ingredients dNTPs,primer,Taq DNA polymerase and Mg^2＋ should achieve 0.1 mmol/L,0.1 μmol/L,0.01 U/μL, 1.48 mmol/ L in PCR reaction system.