Study On Technology Of Detecting Grapevine Fanleaf Virus And Grapevine Leafroll-associated Virus Type Ⅲ By RT-PCR

The techniques of detecting grapevine fanleaf virus(GFLV) and grapevine leafroll-associated virus type III (GLRaVIII) by RT-PCR were studied. First, extract total RNA from grapevine tisste, then, the cDNA of GFLV and GLRaV III coat protein gene were synthesized with reverse transcriptase using 3' primer, and were amplified by PCR using two primers. At last, the fragments of PCR products were cloned, sequenced and analysised. In this study, a series of methods of extracting total RNA were tried,compared and modified, a simple rapid and useful method was developed and a procedure of detecting grapevine virus was set up.The results are as following:1. The quality of RNA is the base the process of RT-PCR,we need pure and undegradated RNA. Phenolic compounds, polysacchrides, proteins and unidentified secondary metabolites are the main factors interfering the isolation of RNA and virus detection. Three methods(SDS, CTAB, centrifugation step by step) were compared and modified, and the last method was perfect for RT-PCR. The extracting buffer is phosphate-buffer, consists of 0. 1% ME, 0. 15g BSA/100M1, 1% PVP,and 0.53gVc/100mL, 0. 14 mol/L NaCl. In this way, we can eliminate the inhibeterant substance which I related and obtained high quality RNA within 3 hours.It indicated that the method is simple, convinent and practical.2. The necessary GFLV and GLRaV â…¢ nucleotide sequences for PCR primers have been published. The 5' and 3' primers of GFLV and GLRaV III coat protein gene were synthesized according to the published.The reverse transcription step was performed with total RNA extracted from infected grapevine tissue. From the synthesized first cDNA strand, 722 bp and 300 bp nucleotide fragments were amplified by two primers, which are the same as what we had expected.3. To check the specificity and efficiency of GFLV and GLRaV â…¢ cDNAfragments amplified, the fragments were isolated from agarose gels and ligated to pGEM-T, transfect E.coli JM 109,the -galactosidase and ampicillin-resistant recombinant plasmids were obtained, these clones were further characterized by restriction enzyme digestion and PCR for the presence of the insert PCR products.The recombinant plasmids were then sequenced by the dideoxy chain termination method. The results showed that there is 99.3% between what we obtained by RT-PCR and the published. So, we consider that the technique of RT-PCR is exact and reliable, and it will give us an important preparation for virus diagnosis and quarantine on grapevine in the future.