A蛋白斑点免疫金染色和双抗体夹心酶联检测齿兰环斑病毒的比较

A蛋白斑点免疫金染色检测齿兰环斑病毒提纯病毒的灵敏度为1.56 ng/μl,检测病汁液的稀释倍数为640倍.同时采用碱性磷酸酯酶标记抗体IgG.IgG包被酶联板的浓度为1 μg/ml,酶标抗体以1 /1000稀释使用.DAS-ELISA技术检测ORSV提纯病毒的灵敏度为0.048 ng/μl,检测病汁液的稀释倍数为20480倍.     

应用免疫金探针定位和定量测定稻种中的水稻细菌性条斑病菌

本文首次报道用免疫金染色和免疫金银染色对水稻细菌性条斑病病种的带菌部位及带菌量的研究。结果表明:病原细菌分布于谷壳、籽实皮、胚和胚乳,主要集中在谷壳上。谷壳外表带菌量多于内表面。籽实皮外表面由于受谷壳污染较之内表面的带菌量多。籽实皮内表面、胚和胚乳的带菌量均较少,差异不显著。谷壳、谷壳内表面、籽实皮、胚乳(或胚)的带菌量之比为36:13:9:1。     

应用免疫金探针定位和定量测定稻种中的水稻细菌性条斑病菌

 本文首次报道用免疫金染色和免疫金银染色对水稻细菌性条斑病病种的带菌部位及带菌量的研究。结果表明:病原细菌分布于谷壳、籽实皮、胚和胚乳,主要集中在谷壳上。谷壳外表带菌量多于内表面。籽实皮外表面由于受谷壳污染较之内表面的带菌量多。籽实皮内表面、胚和胚乳的带菌量均较少,差异不显著。谷壳、谷壳内表面、籽实皮、胚乳(或胚)的带菌量之比为36:13:9:1。     

免疫金银染色技术及其在植物病原细菌研究中的应用

免疫金银染色技术及其在植物病原细菌研究中的应用冯家望莫晓凤（拱北动植物检疫局５１９０２０）自从１９７１年Ｆａｕｌｋ和Ｔａｙｌｏｒ〔１〕首次报道用胶体金标记抗体研究细胞表面抗原以来，免疫金银染色（ｌｍｍｕｎｏ－ｇｏｌｄｓｔａｉｎｉｎｇ）技术已被成功地应...     

免疫凝聚试纸条和TaqMan探针实时荧光PCR检测西瓜细菌性果斑病菌比较研究

 以西瓜细菌性果斑病菌(Acidovorax avenae subsp.citrulli)菌悬液和田间采集的病组织为试材,研究了免疫凝聚试纸条和实时荧光PCR技术检测的灵敏度和适应性。结果表明,免疫凝聚试纸条检测灵敏度为10⁶ cfu/mL,具有简便、快速、易操作特点,适用于田间快速检测和病害诊断;TaqMan探针实时荧光PCR检测灵敏度达10^3~4 cfu/mL,比传统PCR检测灵敏度(10⁵ cfu/mL)提高了10~100倍,且不需要琼脂糖凝胶电泳、溴化乙锭染色和Southern杂交。但需要昂贵的仪器和试剂,适用于室内检测及相关研究。     

应用免疫胶体金银染色技术定位玉米内生细菌

 本文利用光镜免疫胶体金银染色技术对玉米内生细菌进行了定位研究。用分离、筛选于玉米体内具有生防潜力的优势菌枯草芽孢杆菌B20-006菌株的特异性蛋白,制备兔抗血清作为一抗,二抗为金标记羊抗兔IgG,30%甘油-1%戊二醛固定组织,进行冷冻切片免疫胶体金银染色光镜观察。结果表明:枯草芽孢杆菌液浸根处理后5~7d (三叶期)的组织切片中菌体有大量金银颗粒沉淀,内生细菌在根、茎、叶均有分布,大多寄生在植物组织的细胞间隙,菌量根部大于茎和叶部,根、茎、叶的菌量之比为12:3:1,证明枯草芽孢杆菌B20-006菌株为玉米内生细菌,且可在玉米体内繁殖和传导。     

两种免疫胶体金快速诊断技术简介

植物病毒的血清学检测 ,以酶联免疫吸附试验 (ＥＬＩＳＡ)最为普遍和有效 ,它在方法学上比较完善 ,是一种经典的方法。通常ＥＬＩＳＡ反应是在聚苯乙烯反应板上进行的 ,当固相载体变成硝酸纤维素膜时 ,则出现了免疫酶斑点试验 (ＩｍｍｕｎｏｅｎｚｙｍｅＳｐｏｔＡｓｓａｙ ,ＩＥＳＡ)。若将此处的标记物由酶标记改为金标记时 ,因不需要酶促反应的底物 ,缩短了整个检测的时间 ,随着技术的不断进步 ,出现了两种免疫胶体金快速诊断技术即 :斑点免疫金渗滤试验和胶体金免疫层析试验。现分别予以介绍。1　斑点免疫金渗滤试验 (ＤｏｔＩｍｍｕｎ…     

氰烯菌酯胶体金免疫层析方法的建立与验证

氰烯菌酯是我国自主研发的防治小麦赤霉病的新型杀菌剂，国内外尚无类似产品，其残留免疫检测方法亦无报道。本研究通过设计合成半抗原，制备了该农药的鼠源特异性单克隆抗体，通过纳米金标记建立了胶体金免疫层析检测方法。鉴定结果表明：所获得的单克隆抗体类型为IgG1。间接竞争酶联免疫吸附分析(ic-ELISA)结果表明：在38.72～438.08 ng/mL线性范围内，该单克隆抗体对氰烯菌酯的检测灵敏度即20%抑制浓度(IC₂₀)为38.72 ng/mL,50%抑制浓度(IC₅₀)为108.42 ng/mL，与结构类似物杀菌剂嘧菌酯、吡唑醚菌酯、氟醚菌酰胺和啶氧菌酯的交叉反应率(CR)<0.01%，表明该抗体对氰烯菌酯具有较高的特异性。胶体金免疫检测试纸条裸眼观察检出限为50 ng/mL,15 min内可完成检测，与甲氧基丙烯酸酯类杀菌剂无交叉反应。以添加氰烯菌酯的面粉样本进行方法验证，胶体金免疫层析试纸条测试结果与液相色谱-串联质谱(LC-MS/MS)方法的检测结果一致，表明研发的胶体金免疫层析方法可实现样品中氰烯菌酯残留的快速定性以及半定量分析。     

种传番茄溃疡病菌直接PCR和免疫捕捉PCR检测方法之比较

用PBS和种子研磨后的普通病原提取液稀释番茄溃疡病菌纯菌液,并以梯度纯菌液和带菌种子提取液作为模板进行直接PCR和免疫捕捉PCR,比较2种方法在纯菌液和带菌种子提取液中的检测灵敏度,找到一种高特异性、高灵敏度、简便快捷的方法用于检测番茄种子携带的番茄溃疡病菌。结果显示在纯菌液中直接PCR灵敏度为104cfu/mL,免疫捕捉PCR为102cfu/mL;在带菌种子提取液中直接PCR灵敏度为106cfu/mL,免疫捕捉PCR为104cfu/mL;免疫捕捉PCR比直接PCR灵敏度高100倍,尤其在实际种子检测中优势更明显,而且检出时间短,重复性好。     

受侵染细胞内病毒抗原的免疫标记

采用金颗粒免疫标记技术,通过电镜现察,初步明确了豇豆裉绿斑驳病毒(CCMV)在浸染豇豆后,侵染点细胞的变化,病毒增殖及扩散的部位等,并对检测技术提供了今后的改进意见.     

Pre- and post-embedding immunogold labeling and electron microscopy in plant host tissues of three antigenically unrelated MLOs: primula yellows,tomato big bud and bermudagrass white leaf

Methods are described for pre- and post-embedding immunogold labeling of mycoplasmalike organisms (MLOs) in thin sections of infected plants. Antisera against primula yellows (PY), tomato big bud (TBB) and bermudagrass white leaf (BGWL) MLOs, and a monoclonal antibody (mab) against PY were tested with the three serologically unrelated MLOs. Labeling was specific for each MLO and was localized to the outer surface of the MLOs. The antisera performed well in both pre- and post-embedding experiments; the mab reacted well in pre-embedding conditions but gave no labeling with post-embedding. Glutaraldehyde fixation reduced levels of labeling in post-embedding conditions. The results show that these techniques can be used to differentiate MLOs reliably, and extend the usefulness of electron microscopy in this area.     

Localization of potato leafroll virus in leaves of secondarily-infected potato plants

Potato leafroll virus (PLRV) antigen was localized by immunogold labelling in semi-thin leaf sections of secondarily-infected potato plants cv. Bintje. Viral antigen was present in all cell types of the phloem tissue. but occurred most abundantly in the companion cells. Detectable amounts of PLRV antigen were found only in the sieve elements in veins with a large number of infected companion cells. Occasionally, parenchyma cells were also found to be infected. PLRV was not exclusively limited to the phloem tissue in the infected potato plants, but was also found in mesophyll cells neighbouring minor phloem vessles. Spread of virus from cell to cell in the mesophyll was not observed. The distribution of PLRV in the potato leaf tissue has implication on its availability, for acquisition by aphids.     

病毒杀虫剂产品中多角体的染色计数方法

本文介绍了一种直接检测病毒杀虫剂产品中多角体数量的染色计数方法。该染色计数法测得的多角体数量要低于血球计数板的计数结果,但变异系数较血球计数板计数法显著较小,结果稳定,适用于病毒杀虫剂的质量检测。     

Non-structural plum pox potyvirus proteins detected by immunogold labelling

Plum pox potyvirus (PPV) induces in infected Nicotiana clevelandii cells characteristic crystalline inclusions known as nuclear inclusions (NI) when located in the nucleus and as dense material (Dm) when located in the cytoplasm. Crystalline inclusions contain protease (NIa) and RNA-dependent RNA polymerase (NIb) proteins. It is now well established for all potyviruses that cylindrical inclusions contain CI helicase ATPase protein (Martin et al., 1992). The intracellular location of other non-structural PPV proteins remains unknown. Using Escherichia coli expression vectors, specific antibodies were obtained against P1, P3, 6K2 and NIb PPV proteins for which antibodies were not yet available. As expected, NIb antiserum labelled crystalline inclusions. P1, P3 and 6K2 proteins were present in both types of crystalline inclusions found in the nucleus and in the cytoplasm of PPV-infected leaves of N. clevelandii, suggesting that nuclear inclusions and dense material were composed of the same proteins. This composition is discussed.     

麦二叉蚜传播玉米矮花叶病毒的机制

用胶体金标记法和荧光抗体标记法研究了麦二叉蚜(Schizaphis graminum)传播玉米矮花叶病毒(Maize dwaft mosaic virus,MDMV)的机制。麦二叉蚜传播玉米矮花叶病毒需要辅助成份-蛋白酶(Helper component-proteinase,HC-Pro)的参与。在电镜下观察到HC-Pro可以与MDMV粒子结合。用FITC标记的HC-Pro抗体和MDMV抗体证明,HC-Pro可以直接结合到蚜虫口针上;而MDMV粒子不能直接结合到蚜虫口针,必须在HC-Pro的辅助下才能结合到蚜虫口针上。这为HC-Pro在蚜虫传毒过程中起桥梁作用提供了新的证据。MDMV粒子主要吸附在蚜虫口针的尖端和中间部分。     

6种染色方法对黄瓜霜霉病菌不同发育阶段的染色效果比较

 比较了Calcofluor、苯胺蓝、荧光素钠3种荧光染色方法和曲利苯兰酒精-乳酚酸、考马斯亮兰和苯胺蓝组织透明染色法对黄瓜霜霉病菌不同生长发育阶段的染色效果。结果表明,在一定的染液浓度和pH值条件下,3种荧光染色方法均可进行孢子囊、休止孢、芽管、附着胞、孢囊梗等寄主组织外部菌体的原位染色,其中Calcofluor染色方法更稳定、简捷和易于观察。曲利苯兰组织透明染色法能观察黄瓜霜霉病菌的整个生长发育阶段,但该法更适宜胞间菌丝及吸器的染色。     

FM4-64在植物病原真菌和卵菌的细胞膜及囊泡染色中的应用

 FM4-64,a kind of membrane-selective fluorescent dye,was utilized to stain the membrane structure of Botrytis cinerea and Phytophthora capsici.The effect of FM4-64 on hypha was observed under confocal microscope.The results indicated that the plasma membrane,speta and vesicles of the hyphae could be stained with FM4-64 at the concentrations of 1 to 20 μmol/L.The optimal dye concentration was 6 μmol/L for the both pathogens.At the same concentration of FM4-64,the staining of organelles was in a time-dependent manner and the different organelles were stained in diferrent times.The staining of plasma membrane and vesicles should be completed in 5 minutes,as other organelles can be stained after 10 minutes.