Investigation Of Three Grape Viruses And The Rseearch On Molecular Detection Technology

In recent years, Xinjiang grape industy sprung up everywheremainly with the production of processing grape, But with the planting area has expanded each year, the virus diasease is more common. Therefore, to define the type of grape viruses, strenthen seed quaratine inspection, to elimate pathogens is an important measure to control the spread of grape virus disease occurrence and safety prevention and control from the source, studied and established molecular biology detection technology and effective detoxification treatment technology of significant hazard virus, which provided scientific basis for grape virus early diagnosis, quarantine inspection, epidemic early warning and control of diseses.Through fild investigation of grape disease symptoms, RT-PCR amplification and cloning, sequence analysis, defined the main pathogenic rate of GRSPa V was the highest 51.4%, follwed by GLRa V2 48.6%, the infection rate of GFKV is the smallest 36.1%. The GFKV isolate(GFKV) have sequence similarity of 96.31% and 91.03% with Macedoma isolate(KF594431) and Liaoning isolate(JF927942), respectively, GRSPa V isolate(GRSPa V) from Xinjiang showed 96.59% identity with USA isolate(AY368590), respectively, GLRa V2 isolate(GLRa V2) share 93.78%, 93.21% and 92.91% identity with China isolate(JN860997), Frace isolate(EF012721) and Sichuan(China) isolate(DQ911147), respectively. The detection demonstrated that the three grapevine viruses are widespread distributed in Xinjiang with a significant difference.According to coat protein gene sequences of infecting Xinjiang processing grape major virus GRSPa V, GLRa V2, designing of two virus-specitic primers,established double RT-PCRdetection system which can simultaneousil detect two viruses. The target band size of amplification system of GRSPa V, GLRa V2 was 470 bp, 589 bp, the lowest detection limit of viral nucleic acid was 0.9 pg/ul, sensitivity is similar with singlest RT-PCR.At the same time, the study on harmful viruses GRSPa V, according to the specificity of its GRSPa V CP gene, designed different virus-specific primers and Taq Man probes. After optimization of the reaction system and reaction conditions, established a Taq Man probe real-time fluorescenr quantitative PCR detection system which can detect the virus, the maximum detection sensitivity is 0.0475 fg/ul, higher than conventional RT-PCR over 1000 times.