Detection Of Pepper And Garlic Viruses By Multiplex RT-PCR

Pepper and garlic contain high nutrition and special flavor,which are both vegetable crop and condiment. However, the pepper is easily infected by virus disease,which leads to reduce its yield to 30%-50% even failed. The virus can be gradually accumulated in plant during the asexual reproduction process of garlic and cause genetic depression. So, when producing pepper and cultivating garlic of virus free the viral infeetions or detoxification effect and quality should be detected by rapid and effective methods.In this research,viruses including Tobacco Mosaic Virus, Cucumber Mosaic Virus, Potato Virus Y, Broad Bean Vascula Wilt Virus-2of peppe and Leek yellow stripe virus, Onion yellow dwarf virus, Shallot latent virus and Allexiviruses of garlic were researched. Two different 4-plex RT-PCR detection system were built and the occurrence of viruses of pepper and garlic in Hunan were detected by them.In addition, the type of Allexiviruses from the main origin place of garlic in Hunan was analyzed. The main contents and results are following:Changsha and Liling peppers in Hunan province were used as materials. According to the published nucleotide sequences,several pairs of compation primers specific for CMV? TMV?PVY and BBWV-2 were designed and used to establish the RT-PCR in this assay.The samples contain four kinds of virus were used to establish the 4-plex RT-PCR detection system which can detect CMV (323bp)?TMV (237 bp)?PVY (480bp) and BBWV-2 (1057bp) simultaneously,which contain 10×PCR Buffer(-Mg2+) 2.5?L?50mM Mg2+ 3 ?L?2.5mM dNTP5 ? L?primer (-R?-F) 1 ?L?Taq enzyme 0.5 ?L?cDNA2? L, ddH2O make up to 25 ? L; PCR reaction procedure:94? 5min,94? 30s; 57?40s; 72?2min; Cycle number was 40,72 ?10min.43 pepper samples of Hunan province were detected by the established multiplex RT-PCR system.The results indicated that all the pepper samples were detected by viruses.some samples were infected by one virus and some were mixed infected by several viruses,which proved the system can be used in practice.Changsha and chaling garlics in Hunan province were used as materials. According to the published nucleotide sequences,several pairs of compation primers specific for Allexiviruses?OYDV?LYSV and SLV were designed and used to establish the RT-PCR in this assay.The samples contain four kinds of virus were used to establish the 4-plex RT-PCR detection system which can detect Allexiviruses (180 bp), OYDV (265 bp)?LYSV (404 bp) and SLV (592 bp) simultaneously,which contains 2× PCR mix 12.5 ?L?cDNA2 ? L?primer (-F?-R)1?L, ddH2O makes up to 25 u L; PCR reaction procedure:92? 2min,92? 30s,44? 30s,72? 1min, cycle number was 40,72? 10min.159 garlic samples of Hunan province were detected by the established multiplex RT-PCR system.The results indicated that all the garlic samples were detected by viruses.some samples were infected by one virus and some were mixed infected by several viruses,which proved the system can be used in practice.Sequence and sequence identity analysis of the Allexivirusesisolate of garlic samples about changsha and chaling in Hunan showed that they were GarV-D.