Establishment of multiple RT-PCR detection system for four virus in the cultivars of wine grape

Abstract: 【Objective】Grape is one of the most important fruit crops in the world.Most grape production is for wine making.Grape cultivars are uasually vegetively propagated.Hexi of Gansu province have become a large region for planting grape for wine.Virus diseases have spread widely along with the enlargement of planting area.There are 6 kinds of virus of grape in China, including Grapevine virus A (GVA) , Grapevine leaf roll-associated virus 1 (GLRa V-1) , Grapevine leaf roll-associated virus 2 (GLRa V-2) , Grapevine fleck virus (GFKV) , Graperine fanleaf virus (GFLV) and Grapevine leaf roll-associated virus 4 (GLRa V-4) .A simple, fast and efficient detection technology is necessary for controlling the virus diseases in grape【.Methods】16 leaf samples from 13 grape cultivars were collected from 4 grape production bases in Wuwei, Zhangye and Lanzhou city.All samples were frozen separately in liquid nitrogen and were stored at-80℃for further use.Total RNA of all samples were extracted using modified CTAB method, and c DNA were synthesized using M-MLV First Stand DNA Synthesis Kit (Ta Ka Ra, Bio Inc., Japan) .For each virus, the sequences from different regions were downward from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) .After that, 6 pairs of primers from conservative sequences were designed using on online software program Primer 3 (http://primer3.ut.ee/) .To detect the amplification efficiency and the existence of objective virus, each sample was amplified by simple RT-PCR with 13 annealing temperatures from 48℃to 60℃.Furthermore, multiple RT-PCR systems were established by mixing two or more pairs of primers with same annealing temperature.【Results】16 grape samples could be effectively detected by simple RT-PCR.In the Moga Grape Base (Wuwei) , GVA was positively detected from 3 out of 5 grape samples, both GLRa V-1 and GFKV were detected from 2 samples, respectively.In the Huangtai Grape Manor (Wuwei) , GLRa V-1 was positively discovered from 5 samples out of 6 grape samples, GLRa V-2 was detected from 1 sample and 2 sample, respectively.In the Guofeng Organic Grape Manor (Zhangye) and Moga Grape Base (Lanzhou) , GVA, GLRa V-1 and GFKV were detected from most samples while GFLV and GLRa V-4 were not found.With the annealing temperature of 49℃, virus GVA and GLRa V-2 in grape tissue could be positively detected by primers GVA and GL-Ra V-2, respectively.When the annealing temperature was up to 55℃, GLRa V-1 and GFKV could be detected by GLRa V-1 and GFKV, respectively.When the annealing temperature was 60℃, GFLV and GL-Ra V-4 could be detected by GFLV and GLRa V, respectively.Furthermore, three multiple PCR detection systems were established with integration of primer GVA and GLRa V-2 (system I) , GLRa V-1 and GFKV (system II) , and GFLV and GLRa V-4 (system III) , with annealing temperature of 49℃, 55℃, 60℃, respectively.Within three systems, virus GVA and GLRa V-2 could be positively detected at the same time using system I, virus GLRa V-1 and GFKV using system II.In addition, system III could not positively detect the existence of GFLV and GLRa V-4 in grape tissue.Therefore, we suggested that multiple detection system I and II could be used for virus detection in wine grape.【Conclusion】Two multiple PCR systems with higher sensitivity for virus detection were established.The results of detection indicated that viral diseases in grape were ubiquitous in Hexi region.Control and prevention of the virus disease of wine grape should be taken into the consideration.