Development And Application Of Real Time Quantitative PCR Approach For Garlic Viruses

Garlic (Allium sativum L) belongs to a cultivated species of Liliaceae Allium (Liliacea)(Allium tistalosum), is an important vegetable crop. Garlic has the very high nutritional valueand the sterilization、anti-cancer、lowering blood lipid、anti-aging and other important medicinalvalue. Garlic is usually reproduced asexually by garlic cloves (underground bulb). The long-termasexual reproduction leads to virus infection in vivo transferred and accumulated fromgeneration to generation, and virus diseases increased year by year, and deterioration of garlic.Detection of garlic virus is one of the most important part in the monitoring and control of garlicvirus. Compare with the conventional electron microscopy、serological technique、 nucleic acidhybridization technique and the conventional PCR technology, to detect the identification ofplant virus by real-time fluorescence quantitative PCR technology, with high accuracy andapplication. Therefore, the research on common garlic as research material, anchoring garliclatent virus (GCLV)、garlic latent virus (GLV)、Shallot latent virus (SLV)、onion yellow dwarfvirus (OYDV)、 leek yellow stripe virus (LYSV) and shallot yellow stripe virus (SYSV) CPgene conservative region, were established for the real-time fluorescence quantitative PCRmethod of quantitative detection of garlic virus respectively. At the same time, the application ofthese methods of quantitative detection of a preliminary study on two garlic cultivars in thesetypes of viruses, and the main results are as follows:According to the gene sequences of six virusinfecting garlic, GCLV、 GLV、SLV、OYDV、LYSV and SYSV login in GenBank, designs SYBR Green real-time fluorescencequantitative PCR primers for the detection of the sixvirus. Six pairs of primer target sequenceswere located in the sequence of CP; by sequence comparison, the sequence of CP primersdesigned from conserved regions, comparing with the BLAST in the GenBank, confirm theprimer specificity is high.For quantitative detection of GCLV、 GLV、SLV、 OYDV、LYSV and SYSV, SYBRGreen real-time fluorescence quantitative PCR were set up. Establishing the standard curve, thecurve slope were-3.18、-3.40、-3.32、-3.51、-3.55and-3.35, and the slope values were foundin-3.0~-3.6. Amplification efficiencies were106%、97%、100%、93%、91%and99%. Thedissolution curve has only a single peak, no primer dimer and non-specific amplification; so the slope of the curve, the amplification efficiency and primers are in line with the quantitativerequirements.The sensitivity of detection of GCLV、GLV、SLV、OYDV、LYSV and SYSV by SYBRGreen Ⅰ detectionreal-time fluorescence quantitative PCR were42copies of/50μl、16copiesof/20μl、15copies of/20μl、16copies of/20μl、40copies of/50μl and10copies of/20μlrespectively, and compared with common PCR detection, the sensitivity is100times that ofordinary PCR. Experimental repeatability within group, coefficient of variation were0.2%~1.1%、0.8%~6.6%、0.6%~1.2%、1.8%~3.9%、1.8%~4.4%and0.6%~1.6%, with goodrepeatability.Use the SYBR Green real-time fluorescence quantitative PCR method to detect the virus ofShandong zaotai yihao, and Gansu Longnan chengxian chisuan (QC3). The results showthat Shandong zaotai yihao contains four kinds of virus: garlic latent virus、leek yellow stripevirus、 onion yellow dwarf virus and shallot yellow stripe virus. Virus copy number were103.75、105.37、101.45and105.48. Gansu Longnan chengxian chisuan (QC3) contains three kinds ofvirus: garlic latent virus、leek yellow stripe virus and shallot yellow stripe virus. Virus copynumber were102.89,105.34and106.32. It shows that garlic virus is ubiquitous in garlic varieties,and most of the infection were compound.