Study On In-situ RT-PCR Technology Of Viruses Disease In Grapevine

In situ PCR was a combination of PCR and in situ hybridization, which developed in 90's. It could not only examine low copy nucleic acids in tissue, but also could still identify the cellar origin and location of signals.It has become an important morphological research method. Using In situ RT-PCR to detect virus on fruit tree could help us discover the virus's distribution and transferation channel of virus, provide us new experiment basis for detection precaution of virus. The study has great theoretical and practical significances, as it would not only provide a new experimental proof, but also would give a theoretic proof on fruit production.In the experiment,using general RT-PCR to detect a grape sample which take Grapevine rupestris stem pitting associated with virus, and using about 2㎜ stem tip of this sample which grown by seed to prepare paraffin slice, using cDNA probe labeled by Biotin-11-dUTP to conduct Direct In Situ RT-PCR and Indirect In Situ RT-PCR, and determine the distribution of GRSPaV in stem tip through observing the tissue which examined by immunology.The main contents and conclusions are as follow:1. Prepared paraffin slice which suitable for plant plumule, gained the optimal grads and time about ethanol dehydrate: it is suitable for using stem tip as materials to do In situ PCR that the tissue is fixed in 4% PFA over 1h,dehydrated by ethanol 50%,70%,80%,90%,100% concentration one hour respectively.2. A specific fragment of about 355bp was obtained by general RT-PCR, The specific fragment was recovered, ligated, cloned, sequenced by Shanghai Sangon Biological Engineering Technology Corporation. Then it was aligned by BLAST with the sequence AF026278 published representing a homology of 89%.3. Used cloned plasmid to prepare cDNA probe labeled by Biotin-11-dUTP.The length of cDNA probe is 355bp, and find out the suitable concentration on In situ PT-PCR was found.4. Direct In situ RT-PCR detection system and indirect In situ RT-PCR detection system of GRSPaV was established5. Determined the distribution of GRSPaV in stem tip. Have determined GRSPaV some distribution in stem through observing the tissue examined by immunology in experiment many negative controls. The result showed: in grapevine stem tip, GRSPaV distribute along vascular bundle, and there was less virus in other tissue cell, and there was no virus above 0.2㎜ area.