胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒

DETECTION AND IDENTIFICATION OF PLANT VIRUSES WITH DIFFERENT MORPHOLOGY IN LEAF-SAPS BY COLLCIDALGOLD IMMUNOSORBENT ELECTRON MICROSCOPY

本文报道胶体金免疫电镜技术的建立:铜网捕获病毒后,用其稀释约200倍的同源抗血清修饰处理3～5分钟,羊抗兔IgG-金复合物标记3～5分钟,经磷钨酸负染,电镜下可见病毒粒子周围金颗粒特异性吸附,而背景上非特异性吸附很少。用此技术成功地快速检测和鉴定了病汁液中线状病毒(大麦黄花叶病毒,大麦温和花叶病毒,小麦梭条斑花叶病毒,芜菁花叶病连和马铃薯Y病毒)、棒状病毒(烟草花叶病毒、长叶车前草花叶病毒和土传小麦花叶病毒)以及球状病毒(水稻矮缩病毒、黄瓜花叶病毒和大麦黄矮病毒)。用0.5M PBSpH7.0制备病汁液,可大幅度提高同源抗血清对黄瓜花叶病毒的捕获能力。

Colloidalgold immunosorbent electron microscopy was applied effectivelyfor rapid detection and identification of plant viruses,particularly those ofextremely low concentration or complicative in ection of viruses with sameParticle morphology in leaf saps.The grids,which had trapped viruses,weredecorated with homogenous rabbit antisera at dilution of 200～300 for 3～5minutes,labelled by goat anti rabbit IgG-gold complex at dilution of 50～100for 3～5 minutes and stained by PTA.This procedure had been found to beoptimal for heavy,specific labelling of viruses with a minimum of non-specific background labelling and economical use of reagents and eleven virusesin leaf saps,including filamenous(barley yellow mosaic virus,barley mildmosaic virus,wheat spindle streak mosaic virus,tunip mosaic virus and potatovirus Y),rod(tobacco mosaic virus,riborass mosaic virus and soilbornewheat mosaic virus)and spherical(rice drawf virus,cucumber mosaic virusand barley yellow drawf virus),were rapidly detected and identified.Forcucumber mosaic virus,0.5 M phosphate buffer,pH7.0 for homogenization ofleaf tissue gave a much greater field of virus particles in trapping tests.