实时荧光定量PCR法检测草莓镶脉病毒

Real time fluorescent quantitative PCR for detection of Strawberry vein banding virus

为了建立一种特异、灵敏、快速检测草莓镶脉病毒(Strawberry vein banding virus,SVBV)的SYBR Green-Ⅰ实时荧光定量PCR方法,根据已有的检测SVBV的引物信息,合成引物,对不同草莓品种进行PCR扩增。经过克隆、测序及序列比对得到了感染SVBV的阳性植株,利用此植株PCR扩增产物构建重组质粒作为标准品,优化了PCR反应体系,标准曲线的循环阈值(Ct值)与模板浓度有良好的线性关系(R2=0.999 1);进行了灵敏度和重复性试验,敏感性可达到10~1拷贝·μL^(-1),约为普通PCR的1 000倍,重复性好;并与常规PCR方法进行了比较,成功建立了检测SVBV的实时荧光定量PCR法。用该方法检测了部分草莓品种的DNA样品,结果表明,大部分植株都带有SVBV。该方法具有很高的灵敏性和重复性,可用来快速检测草莓植株是否感染SVBV。

To develop a highly sensitive and specific real-time quantitative PCR method with SYBR Green-Ⅰ to quantify Strawberry vein banding virus （ SVBV） , a pair of primers which were reported to screen strawberry SVBV samples of different cultivars was synthesized. Positive PCR products were cloned and sequenced by TA clone tech-nology. The sequences were blasted with those in NCBI database and strawberry genome in GDR database. Results showed that the cloned sequences showed high levels of identity with Strawberry vein banding virus sequences. This fragment was cloned using pEASY-T5 Zero vector to construct the recombinant plasmid which was used as a standard recombinant plasmid. The reaction system was optimized. The sensitivity and repeatability were evaluated. Leaves of strawberry plants were detected by the SYBR Green-Ⅰreal time fluorescent quantitative PCR assay in contrast to the routine PCR method. The results indicated that the real-time PCR assay with a quantitative standard curve of good linearity （R^2 =0. 9991） was successfully established. The sensitivity of detection limit could reach 101 copies·μL^-1 , and it was about 1000 times of the routine PCR. The real-time PCR assay was evaluated with some strawberry samples collected from field. This method had high sensitivity and stability, and could be used in SVBV infection di-agnosis and investigation.