Study On Technology Of Detecting And Occurance Of GFLV And GLRaV-3

The techniques of detecting grapevine fanleaf virus(GFLV) and grapevine leafroll-associated virus III(GLRa-3) by ELISA were studied. From the selection of negative, positive control and antibody or antiserum dilution times, to the result of detecting in different stage and site, We found an integrated detecting program on detecting GLRaV-3 and GFLV. In addition, we compared DAS-ELISA and PAS-ELISA in detecting GFLV. We found, though DAS-ELISA was simpler than PAS-ELISA in steps, PAS-ELISA is clearer than DAS-ELISA in conclusion.We detected GLRaV-3 by immune capture reverese transcription ploymerase chain (IC-RT-PCR), form capture virus, release RNA, transcripe cDNA to the fragment of about 300bp PCR productions were cloned. To check the specificity and efficiency of GLRaV-3 cDNA fragment amplified, the fragment were isolated from agarose gels and ligated to pGEM-T, transfect E. coli JM109, the β -galactosides 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 methods. The results showed that there is 96.7% between what we obtained by IC-RT-PCR and the published. So we considered that the technique of IC-RT-PCR is exact and reliable and quarantine on grapevine in the future.We compared the effectiveness of double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) in detecting grapevine leafroll associated virus-3 (GLRaV-3) in infected symptomatic and nonsymptomatic grapevines throughout the growing season. Tissuesamples are from apical, middle and basal leaves. We found: DAS-ELISA, IC-RT-PCR and RT-PCR detected GLRaV-3 in nearly all samples of basal leaves collected throughout the growing season. In contrast, detection of GLRaV-3 in nonsymptomatic vines was erratic regardless of the detection method that was used, and the results were affected on season and tissue samples. Based on the results, we recommend that the phloem of canes from vines or from dormant cutting be used. Moreover, we thought RT-PCR could detect the fewer viruses than DAS-ELISA and IC-RT-PCR, but it was difficult to extract the RNA from the grapevine tissue, and the result was affected by protein, DNA etc from the plant tissue. IC-RT-PCR and RT-PCR were more sensitive than DAS-ELISA. In additional, IC-RT-PCR was faster and more effective detecting GLRaV-3.