李痘病毒单克隆抗体及TAS-ELISA试剂盒的研制

将纯化的李痘病毒(Plum pox virus,PPV)制剂免疫BALB/c小鼠,用SP2/0骨髓瘤细胞与经李痘病毒免疫的BALB/c小鼠的脾细胞融合,有限稀释法克隆和间接ELISA法筛选出2株稳定分泌李痘病毒单克隆抗体的杂交瘤细胞株3F1,7A8。用间接ELISA方法对所获得的2个杂交瘤细胞株进行亚型鉴定分别为IgG1、IgG3。间接ELISA方法测定腹水效价分别为3F1:1.0×106,7A8:1.0×105。以多克隆抗体为包被抗体、单克隆抗体为检测抗体的TAS-ELISA试剂盒与李痘病毒的D株系、M株系的病毒分离物均有反应,与同属的马铃薯A病毒、莴苣花叶病毒、西瓜花叶病毒2号、马铃薯Y病毒坏死株系不发生交叉反应。     

马铃薯Y病毒云南昭通烟草分离物的检测及年度间差异比较

利用DAS-ELISA检测试剂盒检测昭通烟草脉斑病样品,结果表明存在马铃薯Y病毒O株系和马铃薯Y病毒N株系两个不同株系病毒。利用Sprimer和M4引物扩增、克隆、测序,得到长度为1 771 bp目的片段,该片段包含病毒的外壳蛋白基因序列、3′ UTR序列以及部分Nib基因序列;序列分析表明与湖南HN 2分离物（GenBank No. GQ200836）和美国NE 11分离物（GenBank No. DQ157180）核苷酸序列相似性均为98%,系统进化分析表明其具有较近的亲缘关系。     

北京地区辣椒病毒病毒原种类及黄瓜花叶病毒株系鉴定

近十年来,经鉴定,明确了北京地区辣椒病毒病毒原有7种,即黄瓜花叶病毒(CMV)占病株样品的55％,烟草花叶病毒(TMV)占26％,马铃薯Y病毒(PXY)占13.2％,烟草蚀纹病毒(TEV)占11.8％,马铃薯X病毒(PVX)占10.4％,苜蓿花叶病毒(AMV)占2％,及蚕豆萎蔫病毒(BBWV)占1.4％。其中CMV可划分为4个株系,即重花叶株系,坏死株系,轻花叶株系和带状株系。     

北京地区辣椒病毒病毒原种类及黄瓜花叶病毒株系鉴定

近十年来,经鉴定,明确了北京地区辣椒病毒病毒原有7种,即黄瓜花叶病毒(CMV)占病株样品的55％,烟草花叶病毒(TMV)占26％,马铃薯Y病毒(PXY)占13.2％,烟草蚀纹病毒(TEV)占11.8％,马铃薯X病毒(PVX)占10.4％,苜蓿花叶病毒(AMV)占2％,及蚕豆萎蔫病毒(BBWV)占1.4％。其中CMV可划分为4个株系,即重花叶株系,坏死株系,轻花叶株系和带状株系。     

棉花黄萎病病原菌大丽轮枝菌的快速分子检测

大丽轮枝菌Verticillium dahliae是引起棉花黄萎病的土传病害病原真菌。快速及时地检测出大丽轮枝菌,对棉花黄萎病的早期预警及后期防治具有重要意义。采用聚合酶链式反应(polymerase chain reaction,PCR)检测技术对已报道大丽轮枝菌的检测引物进行验证、筛选和改进,获得1对改进的特异性PCR引物VDS-F/VDS-R。在优化的反应体系与扩增条件下,能特异性地从大丽轮枝菌基因组扩增出l条约520 bp的产物条带,检测灵敏度达到10~(-2)ng/μL;利用该引物可特异性地从含有大丽轮枝菌的土壤及棉花植株组织中检测出病原菌;采用巢氏PCR法对人工病土的检测灵敏度达到了10个孢子/g土。表明本引物的PCR检测体系可用于棉花黄萎病的早期快速检测。     

番茄抗黄化曲叶病基因Ty-2的分子标记及种质资源初步鉴定

以抗番茄黄化曲叶病毒病材料‘CLN2498D’为父本,感病材料‘早粉2号’为母本,以其F2代为研究对象,采用AFLP及SSR两种标记方法,筛选与Ty-2基因连锁的标记。通过对256对AFLP引物及30对SSR引物的筛选,共获得5个AFLP标记和2个SSR标记与Ty-2基因连锁,其中标记E02M11距离目标基因5.8cM,另有3个标记距离均在10cM以内。将标记E02M11用于30份番茄材料的种质资源筛选,获得12个含有该标记的番茄材料,为番茄抗黄化曲叶病毒育种工作提供基础。     

菠菜中3种新烟碱类杀虫剂多残留分析方法研究

寄生隐丛赤壳菌是引起板栗疫病的致病菌。为建立该菌的分子检测技术,本研究首先采用通用引物 ITS1／ITS4对分离自四川雅安、泸州及重庆的寄生隐丛赤壳菌及其他参试菌株的 ITS 区进行 PCR 扩增和测序比对。根据该片段与 GenBank 中隐丛赤壳属其他种的 ITS 序列差异,设计了寄生隐丛赤壳菌的特异性引物 ITSP1／ITSP2,片段扩增大小为462 bp。利用该引物对菌株基因组 DNA 进行扩增,可以将寄生隐丛赤壳菌与其他参试菌区分开,检测灵敏度达30 pg。而以引物 ITS1／ITS4和 ITSP1／ITSP2进行的巢氏 PCR,可检测到30 fg 基因组 DNA,其灵敏度较常规 PCR 提高了1000倍。利用巢氏 PCR 检测体系对发病程度不同的组织和携菌组织进行检测,均能快速稳定地检测出寄生隐丛赤壳菌。     

黄瓜花叶病毒2个分离物的亚组鉴定及株系分化研究

 基于黄瓜花叶病毒甜菜分离物CMV-XJ1和CMV-XJ2独特的生物学特性,利用RT-PCR对接种寄主植物进行了检测。RT-PCR产物的RFLP结果显示,2个病毒分离物的MspⅠ酶切图谱与CMV亚组Ⅰ病毒的酶切图谱很相似,但经EcoRⅠ酶切后出现显著差异;进一步对2个分离物的外壳蛋白基因进行了克隆,序列分析表明,CMV-XJ1和CMV-XJ2归属CMVIB亚组,二者存在着株系分化的趋势。     

应用特异引物和简并引物检测百合斑驳病毒

应用特异引物和简并引物对百合斑驳病毒(LMoV)进行RT-PCR检测.结果表明,特异引物和简并引物均能从LMoV感病百合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,简并引物还能从感染TBV的郁金香病叶中扩增出目标片段.将感染LMoV组织总RNA以10倍梯度稀释成不同浓度检测,特异引物检测的灵敏度为10-4,简并引物的灵敏度为10-3.对特异引物的PCR产物进行克隆和测序,序列分析表明与LMoV不同分离物的序列同源性为90%～99%,说明采用本研究确定的方法检测百合斑驳病毒结果准确可靠.     

甜樱桃李矮缩病毒(PDV) RT-PCR检测方法的优化与应用

以甜樱桃(Prunus avium L.)品种红灯的叶片为材料,提取总RNA,选用随机六聚体引物进行反转录合成cDNA,根据李矮缩病毒外壳蛋白基因设计2对特异引物,分别从感病样品中扩增出与预期片段大小相符的目的片段.通过对RT-PCR反应体系中引物、模板浓度和退火温度的优化,改进了现有的李矮缩病毒的RT-PCR检测方法,并成功用于山东泰安地区甜樱桃果园的病毒调查.另外,还可以扩增18 sRNA,实现对李矮缩病毒外壳蛋白基因表达的相对定量分析.     

Detection of plum pox potyvirus and analysis of its molecular variability using immunocapture-PCR1

We have developed an immunocapture-PCR (IC-PCR) detection technique for plum pox potyvirus (PPV) which is both simple and highly sensitive. This single-day assay can detect about 2000 virus particles (200 fg of virus) diluted in 100 μl of crude plant sap, which is equivalent to a sensitivity about 2000 times better than that of a standard ELISA assay. RFLP analysis and sequencing of the amplified cDNA fragment indicate that three groups of strains with limited intra-group variability can be discriminated. Two of these groups correspond to the previously described D and M serotypes of PPV. The third group contains, so far, only the El Amar Egyptian isolate. Strains belonging to the D or M serotypes can easily be discriminated by Rsal polymorphism in the amplified cDNA fragment. Synthetic oligonucleotides allowing specific amplification of PPV strains belonging either to the D or to the M serotypes have also be designed.     

Potential role of almond in sharka epidemics: susceptibility under controlled conditions to the main types of plum pox potyvirus and survey for natural infections in France 1

The changes in cropping conditions of almond and the development of efficient tools for the detection and characterization of plum pox potyvirus (PPV) populations have led us to reassess the potential susceptibility of this species to sharka disease and its role as a virus reservoir. The susceptibility of almond cv. Aï to nine isolates, representative of the known diversity among PPV populations, was assessed under controlled conditions. Most isolates were able to infect almond, by graft or aphid inoculation, causing generalized stable infections without any obvious sharka symptoms. These infected almonds were found to constitute a potential source of virus for aphid vectors, mainly in the case of M isolates. Surveys were carried out in the south of France, in foci of M and D strains of PPV, to evaluate the presence of natural infections of almond. No typical symptoms were observed and the virus was never detected. It can be assumed that the actual limited prevalence of PPV in France does not lead to a sufficiently high inoculum pressure to allow almond trees to be infected.     

Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp

ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants.     

Production and characterization of a monoclonal antibody specific to the M serotype of plum pox potyvirus

A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.     

Evaluation of peach and nectarine cultivars in Bulgaria for their resistance to plum pox potyvirus

In spring and summer 1989, it was established that plum pox potyvirus (PPV) was present in certain peach cultivars in Bulgaria. At the same time, we started to investigate the distribution of PPV in naturally infected 4–5 year-old peach and nectarine cultivars and hybrids in order to optimize PPV detection. Over 160 peach and nectarine cultivars and hybrids were evaluated. In about 40% of the genotypes, typical plum pox symptoms were observed. The latter were estimated and divided into groups depending on their susceptibility to PPV. Observations were made on the population density of seven aphid species established in the peach orchards. Five proved to be vectors of the virus. Myzus persicae was the vector that played the main role in spreading the virus on peach.     

Susceptibility of apricot cultivars to artificial inoculation with plum pox potyvirus and use of ELISA for its evaluation1

The susceptibility to plum pox potyvirus (PPV) of 13 apricot cultivars and two hybrids was studied under isolated conditions in the experimental orchard of the Research Breeding Station at Vesele from 1989 to 1993 after artificial inoculation of the trees. The plants were inoculated in the nursery in 1988 by chip-budding with a local source of virus and then planted into the field trial. First symptoms of plum pox appeared on the leaves in 1990. Reinoculation of symptomless trees was done in summer 1991. By 1993, plum pox had totally invaded some apricot trees of the most susceptible cultivars (Madarska, Velkopavlovicka, Kisinevskij rannij, Ligeti orias, Bergeron, Vesna and Vestar). Uninoculated control trees remained symptomless despite the proximity of diseased trees and the fact that the orchard was not sprayed with insecticides during the period of experiment. Cvs Julskij, Veecot, Vegama and Veharda remained free from plum pox symptoms on leaves and fruits. In 1993-05/06, the trees were assayed for the presence of the PPV antigen by ELISA. Leaf samples from 33 trees showing symptoms were positive. Samples prepared from the healthy tissues of leaves with symptoms were negative. Other trees (indexed by ELISA) growing in the neighbourhood of diseased trees in the period of 5 years were visually and ELISA negative.     

Serological characterization of Italian isolates of plum pox potyvirus1

An Italian isolate of plum pox potyvirus (PPV) from apricot, Ispave 17, was used as antigen for production of monoclonal antibodies. Six clones secreting specific antibodies to PPV were obtained. All these monoclonal antibodies were used to test a collection of different Italian PPV isolates, collected from plum, apricot and peach orchards, and other European isolates (including PPV-D and PPV-M serotypes), using DAS-ELISA, SDS-PAGE, western blot and GIEM. In western blot analysis, the PPV-M and PPV-D coat protein, detected directly from crude peach GF305 extracts, showed different electrophoretic mobility, the coat protein of PPV-M being slightly larger than that of PPV-D. ELISA tests, performed with fixed dilutions of antibodies and limiting dilutions of clarified samples, showed with some monoclonal antibodies a marked difference between PPV-M and PPV-D strains, at ratios greater than 1:40 (w/v). Also in GIEM some monoclonal antibodies gave a good labelling reaction only with PPV-D serotype. With the help of this differentiation, it was found that all Italian isolates tested were of the D serotype and none of the severe M strain of PPV, which has not been reported in Italy.