关键词: 草莓镶脉病毒, SYBR GreenⅠ, 实时荧光定量PCR

Abstract: 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 technology. 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Ⅰ real 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 （R2 =0.999 1） was successfully established. The sensitivity of detection limit could reach 101 copies·μL-1， and it was about 1 000 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 diagnosis and investigation.

Key words: Strawberry vein banding virus；SYBR Green Ⅰ, realtime fluorescent quantitative PCR