Studies On The Molecular Detection Reagent Kit Of Grapevine Virus B

Grapevine Corky Bark (GCB) is a disease that caused by Grapevine Virus B (GVB), which made a great harm to grapevine species and cultivars. Once the virus infected its host, it would exist in the host all the time. GVB caused a slow decline in the growth and output of grapevine, as well as the fruit character. After several years, GVB would make a major impact on grapevine growth and yield, fruit becoming macrocephalic and inedible, losing its commercial value. Finally, it would damage the tree to death. It can't be estimated to the economic loses caused by GVB. But, there hasn't an effective medication that could prevent or cure GVB so far. At present, the only and feasible measure is quarantine and controlling its diffusing. The purpose of this study is to establish the rapid, efficient and specific reagent kit to detect GVB. Reagent kit to detect GVB has become a precondition to the quarantine of young plant and identifying to resistance early and become a guarantee to virus-free culture in grapevine. So the detection of GVB is very significative in application.Grapevine plants in years at Shihezi were selected as materials. The study had gained RNA isolated from phloem of grapevine. Firstly the two-step RT-PCR detection system has been established, and then the sequenced results have been aligned with that published in NCBI. Using cloned plasmid the nucleotide acid spot hybridization (NASH) detection of GVB has been established by detecting 19 samples. Then the optimized one step RT-PCR and NASH detection system of GVB have been established, by which the reagent kit has been assembled.The study includes five major conclusions as followed:1. The modified SDS method was used in this study. The total RNA with a high concentration extracted by the modified method had the clearer bands and can be stored for a longer time.2. A specific fragment of about 460bp was obtained by RT-PCR using the templates of total RNA and specific primer. The specific fragment was recovered, ligated, and then transformed into E.coli XL1. After blue-white selection, activation of bacteria and isolation of plasmid, it was sequenced by Shanghai Sangon Biological Engineering Technology Corporation. Then it was aligned by BLAST with the sequence X75448 published representing a homology of 81%.3. The biotin-11-dUTP labeled cDNA probe prepared by cloned plasmid was used for detection of 19 samples by nucleotide acid spot hybridization (NASH) system established in this experiment. The result was the same as that detected by two-step PT-PCR.4. The optimized One-step PT-PCR and NASH detection system of GVB was established and then assembled into reagent kit.5. It was proved that this reagent kit had good stability and specificity by the detection of 19 samples.