珠葱中葱潜隐病毒SYBR GreenⅠ实时荧光RT-PCR检测方法的建立与应用  

Establishment and Application of a SYBR GreenⅠReal-time Fluorescent RT-PCR for Detecting Shallot latent virus on Shallot(Allium cepa var.aggregatum)

作　　者：刘悦 刘建青 张春雨[1] 苏颖[1] 李小宇[1] 王永志[1] LIU Yue;LIU Jian-Qing;ZHANG Chun-Yu;SU YingLI;Xiao-Yu;WANG Yong-Zhi(Jilin Academy of Agricultural Sciences,Gongzhuling 136100,China;College of Life Sciences,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林省农业科学院,公主岭136100 [2]吉林农业大学生命科学学院,长春130118

出　　处：《农业生物技术学报》2023年第3期659-666,共8页Journal of Agricultural Biotechnology

基　　金：吉林省农业科技创新工程(CXGC2021ZY011);吉林省科技厅重点科技研发项目(20230202108NC);国家重点研发计划子课题(2017YFD0201604)。

摘　　要：葱潜隐病毒(Shallot latent virus,SLV)是危害珠葱(Allium cepa var.aggregatum)等葱属植物的主要病毒之一,在葱属植物种植区普遍存在。本研究根据GenBank中已登录的SLV外壳蛋白(coat protein,CP)基因保守区,设计并合成1对特异性引物,通过对引物浓度和退火温度进行优化,建立了基于SYBR GreenⅠ的SLV实时荧光RT-PCR(reverse transcription PCR)检测方法。该方法标准曲线Ct值与模板浓度的对数呈良好的线性关系,扩增效率为94.529%,相关系数R2为0.9996;与胡葱黄条病毒(Shallot yellow stripe virus,SYSV)、洋葱黄矮病毒(Onion yellow dwarf virus,OYDV)和青葱X病毒(Shallot virus X,SVX)均无交叉反应;最低检出限为10^(-7)倍稀释cDNA,为常规RT-PCR的1000倍;组内和组间变异系数分别为0.02%~0.63%和0.03%~1.24%,表明该方法稳定性好。利用该方法对珠葱叶和鳞茎的SLV含量进行测定,并对50份疑似SLV感染的珠葱样品进行检测,结果表明,珠葱叶中SLV含量明显高于鳞茎,且SYBR GreenⅠ实时荧光RT-PCR检出46份阳性样品,检出率较常规RT-PCR高6%。本研究基于SYBR GreenⅠ建立的SLV实时荧光RT-PCR方法为珠葱SLV的快速检测提供技术支持。Shallot latent virus(SLV)is one of the major viruses that damage shallot(Allium cepa var.aggregatum)and other Allium crops and prevails in many Allium-growing regions.To develop a SYBR GreenⅠreal-time fluorescent RT-PCR assay for the sensitive detection of SLV,a pair of specific primers were designed and synthesized based on the conserved region of SLV coat protein(CP)gene registered in GenBank and a series of optimization including primers concentration and annealing temperature were performed.Ct values were linear with the logarithm of the template concentration from standard curve of cDNA.The amplification efficiency was 94.529%and the correlation coefficient was 0.9996.There was no crossing reaction with Shallot yellow stripe virus(SYSV),Onion yellow dwarf virus(OYDV)and Shallot virus X(SVX).The lowest detection limit was 10-7 dilution fold,which was 1000 times higher than that of routine RT-PCR.The coefficients of variation of intra-assay and inter-assay were 0.02%~0.63%and 0.03%~1.24%,respectively,indicating the excellent stability of the method.The SYBR GreenⅠreal-time fluorescent RT-PCR was used to determine the content of SLV in shallot leaves and bulbs and to detect 50 samples from suspected infectious shallot.The results showed that the content of SLV in leaves was significantly higher than that in bulbs,and 46 out of 50 samples were detected to be positive by this assay,and the detection rate was 6%higher than that of conventional RT-PCR.Therefore,the SYBR GreenⅠreal-time fluorescent RT-PCR method for detection of SLV provides technical support for the rapid detection of SLV on shallot.

关 键 词：珠葱 葱潜隐病毒 SYBR GreenⅠ实时荧光RT-PCR 

分 类 号：S432.1[农业科学—植物病理学]