葡萄扇叶病毒的ELISA检测技术

本文是关于葡萄扇叶病毒ＥＬＩＳＡ检测技术的报告。采用Ｌ．ｐｉｎｋ的方法提纯病毒，制备了兔抗血清和小鼠腹水抗体，效价分别达１：５１２００和１：５×１０￣６，特异性较强。经间接双抗体夹心ＥＬＩＳＡ检测ＧＦＶ提纯病毒最低浓度为３ｎｇ／ｍｌ。对葡萄的根、幼叶和嫩茎抽提液检测结果一致，稀释倍数１：２０。     

葡萄卷叶病毒RT-PCR检测技术研究

以葡萄叶片和皮层为材料,对植物总RNA和dsRNA提取方法进行了研究,从中筛选出了提取葡萄总RNA和dsRNA的适合方法。建立了GLRaV RT-PCR的有效体系,在有效体系基础上,通过研究RT-PCR主要成分对RT-PCR的影响,确立了GLRaVRT-PCR的优化体系。     

葡萄扇叶病毒RT-PCR检测技术研究

以扇叶病株为材料,根据病毒编码外壳蛋白的核苷酸序列,设计特异引物P_1(1,064—1,083),P_2(762—781),建立了以RNA为模板的GFLV的RT-PCR检测体系。PCR产物电泳谱带明显,与预期的引物应扩增321 bp片段完全吻合。并利用建立起来的技术体系,对田间葡萄进行了随机取样检测,取得了很好效果。此方法操作简单,检测周期短,准确率高,具有广泛和更进一步的应用价值。     

葡萄卷叶病毒RT-PCR检测技术研究

以葡萄叶片和皮层为材料,对植物总RNA和dsRNA提取方法进行了研究,从中筛选出了提取葡萄总RNA和dsRNA的适合方法. 建立了GLRaV Ⅲ RT-PCR的有效体系,在有效体系基础上,通过研究RT-PCR主要成分对RT-PCR的影响,确立了GLRaV RT-PCR的优化体系.     

斑点免疫金检测鸡传染性支气管炎病毒的研究

通过胶体金标记提纯后的兔抗ＩＢＶＩｇＧ，建立了一种以微孔滤膜为固相载体，以红色胶体金为标记物，检测鸡传染性支气管炎病毒（ＩＢＶ）的斑点免疫金测定法（ＤＩＧＦＡ）。ＤＩＧＦＡ对ＩＢＶ的最小检出量为２０．３×１０￣（－８）ｇ，在１０份自然病例样品中检出８份。鸡胚培养法以育传３～５代，出现侏儒胚者为阳性，从上述样品中检出６份。对接种ＩＢＶ的鸡胚尿囊液，ＤＩＧＦＡ法检出率为１００％。实验结果表明，ＤＩＧＦＡ法对鸡传染性支气管炎的早期诊断具有简便、灵敏、快速、特异性强和重复性好等优点。     

葡萄卷叶病毒主要检测技术比较

葡萄卷叶病(GLRaV)是葡萄上的一种重要病毒病害。本文主要阐述了该病的病原、分布及近些年来各种检测技术在该病检测中的应用情况,分析了各种检测技术的优势和不足及如何在该病检测中对各种方法做合理的搭配应用,优势互补,提高效率。     

百合病毒检测技术研究进展

笔者对百合病毒的检测技术作了综合评述,主要有植株直接观测法、指示植物接种鉴定法、电镜技术、血清学检测和分子生物学检测技术等,综合应用各项技术使检测更加灵敏、可靠。目前,常用的检测以血清学技术的ELISA为主、分子生物学技术的RT PCR为主。基因芯片检测技术是近几年发展起来高效的检测手段并在植物病毒检测上有广泛的应用前景。     

猪瘟病毒检测技术研究进展

综述了猪瘟病毒检测技术的研究现状,并对今后的研究趋势进行了展望。     

葡萄茎痘伴随病毒cDNA探针检测技术研究

选取经RT-PCR检测确认带葡萄茎痘伴随病毒的全球红葡萄品种,以葡萄叶片、皮层作为试材,采用SDS法提取高质量总RNA作为模板,以GRSPaV的特异互补引物引导,反转录合成cDNA,通过PCR扩增,获得830 bp的预期目的片段,并将其克隆测序,其同源性与NCBI中的相关序列的同源性达96%.利用克隆质粒合成了生物素标记的GRSPaV的cDNA探针,在此基础上,研究了影响GRSPaV斑点杂交的因素,结果表明当探针浓度为200 ng/mL,甲酰胺浓度为45%,42℃杂交反应4h,可获得较高灵敏度.     

Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy

Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated the effects of ribavirin at different concentrations(15 and 25 μg mL~(–1); R15 and R25, respectively), thermotherapy(37°C; T), and the combination of ribavirin and thermotherapy(R15+T and R25+T) on eliminating viruses from grapevine plants in vitro. Both R15 and R25 had phytotoxic effects and weakened plant growth. Thermotherapy positively affected the growth of grapevine plants. Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25. The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively. The survival rates of regenerated plants after all treatments were 68.0%. Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses. The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively. The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively. The elimination rate of GFkV was 23.9% higher in R25(35/44) than in R15(25/45), and that of GRSPaV was 7.0% higher in R25 than in R15. The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.     

香石竹坏死斑点病毒的鉴定与检测

调查表明云南省昆明市团结乡香石竹普遍发生病毒病。电镜负染检测采自团结乡香石竹病毒病病样,病叶汁液中观察到弯曲长线型的病毒粒体,长度约1000~1600 nm,直径约12 nm。对这些病样的叶片进行间接ELISA检测,所有样品与香石竹坏死斑点病毒抗血清都呈阳性反应。按照报道的长线形病毒属简并引物合成引物,采用RT-PCR法对血清反应呈阳性的香石竹总RNA扩增了1059ntsHSP70基因部分编码序列,将其克隆到pMD18-T载体上,并进行序列分析。根据测序结果设计引物建立RT-PCR扩增检测方法并测定其灵敏度。结果表明,使用简并引物和新设计的引物均能从CNFV感病香石竹组织中扩增出与预期大小一致的目标片段,而健康组织无此扩增产物。灵敏性测定结果表明该特异性引物RT-PCR可从稀释106植物总RNA中检测出病毒。     

A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types

To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR) was established. This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR. An excellent linear correlation(R2=0.997) and a high amplification efficiency(E=97.5%) were obtained from the standard curve of this method. Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility. The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types. The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR. The detection rates in spring, summer, autumn and winter increased gradually. Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR. In general, old petioles and branches were the best tissues for GVE detection. The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR. The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR. GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%. A total of 33 out of 363 samples(belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR(9.1%), which was more than twice the rate of the conventional RT-PCR(3.9%).     

甘肃葡萄扇叶病毒和卷叶病毒的检测

采用 DAS-ELISA 检测方法对甘肃地区的葡萄主要品种进行扇叶病毒和卷叶病毒的调查和测定。结果表明,这两种病毒病在甘肃地区的主要葡萄产区均有发生。兰州地区和一些老的葡萄产区葡萄带病毒病较严重。通过对比植株下部枝条的幼叶、幼茎、老叶和老茎病原的检测结果,说明植株下部幼嫩组织是卷叶病毒最适的检测部位。     

烟草种质对气候斑点病抗性的初步鉴定

调查了59个烟草种质田间气候斑点病的自然发病情况，结果表明：国内烤烟品种对烟草气候斑点病抗性明显好于国外引进的烤烟品种，晒烟品种对气候斑点病的抗性好于烤烟品种；花叶病的发生加重了烟草气候斑点病的危害。     

四川葡萄病毒病田间普查及血清学鉴定

对四川葡萄主产区病毒病的发生和危害状况进行田间普查,并采集了48份葡萄枝条样本进行病毒病的血清学检测和鉴定。经双抗体和三抗体夹心酶联免疫法测试后．从采自四川的这批样本中分别检测出了葡萄茎沟病毒(GVA)、葡萄栓皮病毒(GVB)、葡萄扇叶病毒(GFLV)、葡萄斑点病毒(GFkV)、葡萄卷叶病毒-2(GLRaV-2)和葡萄卷叶病毒-3((GLRaV-3)6种危险性的病毒(株系),它们的检出率分别为14.6％、8．3％、10．4％、27.1％、14.6％和20．8％,另外两种靶标病毒(株系)葡萄卷叶病毒-1(GLRaV-1)和南芥菜花叶病毒(ArMV)则未被发现。在四川省内一些老龄葡萄园内．以及部分引进的葡萄新品种上,病毒病的发生和流行极为普遍,并给葡萄的正常生长和生产造成了严重危害。     

生长度日(GDD)对酿酒葡萄霞多丽物候期影响的研究

摘 要：本文通过对宁夏贺兰山东麓产区酿酒葡萄霞多丽（Chardonnay）的4个重要物候期 - 萌芽、盛花、转色和成熟期的观测记录和对应期热量指标生长度日（GDD）的研究,构建了拟合曲线方程并计算出不同物候期的GDD参数,旨在更好地对了解当地的葡萄物候期发生规律与GDD之间的关系,以期达到通过气象预报数据来预测葡萄物候发生时期,指导葡萄园田间操作和科学精准化管理的目标。     

酿酒葡萄区划热量指标的研究

在对目前酿酒葡萄区划热量指标进行总结和分析的基础上,通过对几种热量指标的比较和我国气候特点的分析认为,以无霜期作为我国酿酒葡萄区划的热量指标,即以无霜期≥160 d(30年平均值,且在30年中无霜期<150 d的次数不超过3次)作为我国酿酒葡萄栽培的热量最低限(区划北界)是适宜的,比其他热量指标确定的区划界限更有实际应用价值。     

葡萄枝条中多酚类物质的超声波辅助提取

【目的】探讨利用超声辅助技术从葡萄枝条中提取多酚类物质的最佳工艺条件,并比较8个酿酒葡萄品种枝条中多酚类物质含量的差异。【方法】对影响葡萄枝条中多酚类物质提取率的乙醇溶液体积分数、浸提温度、料液比(质量(g)与体积(mL)比)、提取时间和提取次数进行单因素试验,并在此基础上设计4因素3水平的正交试验,以确定超声波提取的最佳工艺条件;利用最佳工艺条件提取8个葡萄品种枝条中的多酚类物质,并对其含量进行测定。【结果】超声波辅助提取葡萄枝条中多酚类物质的最佳条件为:以体积分数60%乙醇溶液为提取液,料液比为1∶12,超声提取时间为30 min,于20℃下浸提2次。在最佳工艺条件下,多酚类物质的提取率为97.38%。8个酿酒葡萄品种枝条中的多酚类物质含量均大于20 mg/g,其中品丽珠含量最高(32.855 4 mg/g),其次为梅尔诺(31.967 7 mg/g),以8804最低(20.815 6 mg/g)。【结论】确定了超声波辅助提取葡萄枝条中多酚类物质的最佳工艺条件,在此工艺条件下提取率最高可达97.38%。     

双夹心ELISA检测鸡EDS-76病毒抗原的研究

应用鸡抗EDS-76病毒、兔抗EDS-76病毒抗体和羊抗兔抗体,建立了检测EDS-76的双夹心ELISA方法,本试验确定的鸡抗EDS-76病毒抗体、兔抗EDS-76病毒抗体和HRP-羊抗兔抗体的最佳工作浓度分别为1∶160、1∶300和1∶1000。本法对纯化EDS-76病毒的最低检出量约为5 ng/孔。通过对EDS-76病毒试验感染鸡脏器带毒、排毒情况和临床样本的检测,表明双夹心ELISA方法对EDS-76病毒的检测特异性强、敏感度高、适合于成批样本的检测。