Detection Of Three Garlic Viruses By RT-PCR And Multiplex RT-PCR

Garlic is one of the important economic crops in the word.All kinds of diseases and insectpests in the bonds of the whole garlic throughout the growth process, especially called “plantcancer” plant virus disease.It is the threat of garlic production one of main diseases,and seriousinfluence the quality and yield of garlic,since asexual reproduction is the main mode ofproduction of garlic.Cultivate virus-free garlic is the most effective prevention and cure virusdisease method. In the process of cultivating virus-free garlic need to be fast and practicaldetection technology of garlic to identify the type of virus and determine the effect and qualityof the cultivate virus-free garlic.The garlic named“Gansu chengxian chisuan”,“Chengxianzaosuan qishenglinjing”and Shandong “lusuanwang No.2”were chosed for the samples,established multiple RT-PCR system detected of the garlic virus, And optimization multiplepolymerase chain reaction system by the orthogonal design method. The detection techniquewas used to detect occurrence of the two viruses in different garlic cultivars. As a result, thestudy provides technology reference for simultaneously detection of more garlic virus.According to the published nucleotide sequence, three pairs of compation primers specificfor Garlic latent virus (GLV),Leek yellow stripe virus(LYSV) and Onion yellow dwarfvirus(OYDV) were designed in the conserved regions of the coat protein gene in this assay.Single RT-PCR was carried out and three distinct fragment were obtained:849bp(GLV),276bp(LYSV) and571bp(OYDV), which were the same as the designed ones in size. Thesamples contain three kinds of virus were filtered.A multiplex RT-PCR was established for simultaneously detection of GLV, LYSV andOYDV,base on the single RT-PCR.The orthogonal design L16(45) was used to optimizemultiplex PCR system in five factors:Taq DNA polymerase, Mg2+, DNA templet, primer anddNTP, at four levels respectively. All samples infected by GLV, LYSV and OYDVcould beamplified by multiplex RT-PCR, and yielding three special bands of GLV (849bp), LYSV(276bp),and OYDV(571bp).Sequence analysis of the amplified products showed that thesequence identity of GLV was100%compared with DQ520093,and98%with AB004567;LYSVwas93%with D11118and AB194622;OYDV was98%with GQ475358and GQ475360.“Gansu chengxian chisuan”,“Chengxian zaosuan qishenglinjing”and Shandong“lusuanwangNo.2” were detected by the established GLV, LYSV and OYDV multiplex RT-PCR system.Theresult indicated that all the garlic samples were detected by viruses, and77%of the samples weremixed infection of two viruses.The result show that GLV,LYSV and OYDV have infected moresamples and also occur complex infection.